Compositions and methods for treating gluten intolerance and disorders arising therefrom

ABSTRACT

The invention described herein relates to methods and compositions for treatment of gluten intolerance and related conditions (e.g., celiac disease and gluten sensitivity), or inhibition of inflammation and/or immune response in the intestine due to antigenic food peptides, by administration of a pharmaceutical composition comprising one or more  Nepenthes  enzymes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Nos.62/012,865, filed Jun. 16, 2014, and 62/118,396, filed Feb. 19, 2015.The content of each prior application is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

Provided herein are compositions and methods for the treatment of glutenintolerance and related conditions, such as celiac disease or glutensensitivity. Further provided herein are compositions and methods forattenuating or preventing intraepithelial lymphocyte (IEL) infiltrationinduced by the presence of food protein antigens in the intestine. Suchfood protein antigens include difficult to digest proline rich foodssuch as proteins found in wheat, barley, rye, etc. that contain gluten.Gluten, in particular, is partially hydrolyzed in the gastrointestinaltract and can lead to IEL infiltration and production of antibodiesincluding endomysial IgA and anti-tissue transglutaminase. Thecompositions and methods of this invention provide for reduced amountsof such food protein antigens in the intestine which, in turn, reducesthe amount of IEL infiltration of the intestine.

BACKGROUND OF THE INVENTION

Several diseases are mediated by reactions to antigenic food proteins insusceptible individuals. For example, ingestion of wheat, barley, andrye, which contain antigenic food proteins (e.g., gluten) may causeabnormal autoimmune responses, such as celiac disease, wheat allergy anddermatitis herpetiformis, in gluten intolerant individuals. Gluten is amixture of glutamine- and proline-rich glutenin and prolamin proteinmolecules.

Celiac disease is an autoimmune disorder affecting the small intestine.Most of the individuals having the abnormal autoimmune responsescharacteristic of celiac disease express the human leukocyte antigen(HLA) DQ2 or DQ8 molecules. Symptoms of the disease are caused by areaction to gluten proteins, and may also include other storage proteinsin the grain products consumed (e.g. serpins, purinins). Clinically, thedisease is detectable in part through the quantitation of antibodiesspecific for gluten and tissue transglutaminase (tTG). The autoimmuneresponses result in the development of small intestinal mucosal villousatrophy with crypt hyperplasia and mucosal inflammation. Symptoms ofceliac disease can vary from individual to individual, and may includeone or more of fatigue, chronic diarrhea, constipation, malabsorption ofnutrients, weight loss, abdominal distension, anemia, as well as asubstantially enhanced risk for the development of osteoporosis andintestinal malignancies (lymphoma and carcinoma).

Type I diabetes is a risk factor for celiac disease. Autism is alsoassociated with celiac disease, and a gluten-free diet may helpalleviate some symptoms of autism. Similarly, it is believed that somepeople with attention deficit hyperactivity disorder exhibit fewersymptoms when gluten is removed from their diets. Other conditions thatmay benefit from elimination of dietary gluten include rheumatoidarthritis and fibromyalgia.

Treatment for gluten intolerance, especially celiac disease, commonlyinvolves a lifelong, strict gluten-free diet. However, gluten-free dietis inconvenient, restrictive, and gluten is difficult to avoid.Therefore, effective alternative treatments of gluten intolerance andceliac disease are needed.

SUMMARY OF THE INVENTION

This invention relates to the discovery that administration of apharmaceutical composition comprising one or more Nepenthes enzymes asdescribed herein, in combination with a potentially antigenic foodprotein, results in a decrease in immune response to the antigenic foodprotein after ingestion, including a decrease in infiltration and/orproduction of intraepithelial lymphocytes in the intestine.Intraepithelial lymphocytes are T cells that are interspersed betweenepithelial cells of the large and small intestine. An increased T cellcount is an early indicator of inflammation and is potentiallyassociated with gluten intolerance, including celiac disease.

The toxic properties of gluten proteins (e.g., gliadins and glutenins)are believed to be largely due to proline- and glutamine-rich peptidesthat are produced during incomplete degradation of the proteins by humandigestive enzymes (including pepsin). Gastric and pancreaticendoproteases are unable to cleave these toxic or immunogenic peptidebyproducts of incomplete degradation, at least in part due to the factthat such enzymes lack specificity for proline and/or glutamine. Thepeptides are believed to cause numerous intestinal symptoms in sensitiveindividuals, including intraepithelial lymphocytosis, villous atrophy,and/or inflammation. Other proteins present in wheat may also beimplicated in the autoimmune response, including serpins, purinins,alpha-amylase/protease inhibitors, globulins, and farinins.

T cells are a first responder to antigenic insult (i.e., presence oftoxic food peptides) in a sensitive individual. T cells react quickly toantigen insult and cause inflammation and, in some cases, degradation ofthe intestine. A reduction in T cells in the intestine thus indicates adecreased immune response, and is a potential indicator of reduced oreliminated symptoms associated with immunogenic food (e.g., gluten)consumption in sensitive individuals.

Without being bound by theory, it is believed that contacting gluten (orother antigenic protein) with a pharmaceutical composition as describedherein breaks down the protein into small polypeptide fragments thatreduces or eliminates an immune response (i.e., are not toxic or areless toxic).

It is contemplated that a pharmaceutical composition as described hereincan be used to degrade dietary proteins, particularly proline- and/orglutamine-rich proteins, that are not effectively degraded by digestivetract enzymes. It is further contemplated that such degradation wouldincrease absorption of the proteins and/or decrease immunogenicity. Sucha result may have beneficial effects on the symptoms of intestinaldiseases and disorders (e.g., celiac disease, gluten intolerance,irritable bowel syndrome, colitis, Crohn's disease, food allergies andthe like). In one embodiment, administration of the pharmaceuticalcomposition improves nutrient absorption.

The pitcher secretions of Nepenthes, a carnivorous pitcher plantcommonly known as monkey cups in tropical regions, include a number ofproteases. Concentrated Nepenthes pitcher fluid has high specificity forproline- and glutamine-rich gluten peptides. U.S. Patent ApplicationPublication Nos. 2014/0186330 and 2014/0140980, incorporated herein byreference in their entireties, describe the activity and specificity ofconcentrated Nepenthes pitcher fluid and recombinant Nepenthes enzymes.The pitcher fluid is acidic, and the enzymes therein are generally mostactive at acidic pH.

Nepenthesin (EC 3.4.23.12) is an aspartic protease that can be isolatedor concentrated from Nepenthes pitcher secretions, as well as a varietyof other plant sources. Tökés et al., Digestive Enzymes Secreted by theCarnivorous Plant Nepenthes macferlanei L., Planta (Berl.) 119, 39-46(1974). It has been found that the activity of nepenthesin is higherthan that of pepsin (EC 3.4.23.1), an enzyme present in the stomach ofhumans that is partly responsible for degrading food proteins intopeptides. Nepenthesin has two known isotypes: nepenthesin I (known tohave two variants: nepenthesin Ia and nepenthesin Ib) and nepenthesinII.

In one aspect, this invention relates to the discovery of a novel prolylendopeptidase, neprosin, which possesses a high proteolytic activity forcleaving proline-rich proteins and oligopeptides (such as glutenproteins). Neprosin can be isolated or concentrated from the pitchersecretions of Nepenthes, is active at a broad pH range, and isespecially active at low pH (e.g., about 3 to 5). The neprosin proteinsequence is not homologous to any other known protein in the genomicdatabases. Neprosin can efficiently cleave peptides on the carboxy(C)-terminal side of proline. This cleavage appears to be highlyspecific.

Neprosin, nepenthesin I, and nepenthesin II, alone or in combination,are able to cleave toxic food peptides into smaller, non-toxic peptides.Because the enzymes are active at a broad acidic pH range, digestion bythe enzymes can initiate in the acidic environment of the stomach.

This invention is further based on the discovery that such enzymecompositions are capable of degrading food protein antigens to a levelwhere the immune response in the intestine, as measured by IELinfiltration, is attenuated or eliminated when used in combination withfood. IEL infiltration due to the presence of peptidic food antigen(s)is an early biological indicator of sensitivity to food antigen (e.g.,gluten). Accordingly, in one aspect, this invention is directed to amethod for attenuating or preventing an immune response to food proteinantigens in the intestine of a mammal, which method comprisesadministering to the mammal an effective amount of a pharmaceuticalcomposition comprising at least one Nepenthes enzyme. In one embodiment,the at least one Nepenthes enzyme is nepenthesin I, nepenthesin II,neprosin, a variant thereof, or a mixture thereof. In one embodiment,the amount of the pharmaceutical composition is effective to attenuateor prevent IEL infiltration of the intestine due to the presence of thepeptidic food antigen(s). In one embodiment, the IEL infiltration is dueto incomplete digestion of a potentially antigenic food protein byendogenous gastric and/or intestinal enzymes. In one embodiment, thecomposition is administered to the mammal prior to ingestion of apotentially antigenic food or protein. In one embodiment, thecomposition is administered to the mammal with ingestion of apotentially antigenic food or protein. In one embodiment, thecomposition is administered to the mammal after ingestion of apotentially antigenic food or protein. In one embodiment, thecomposition is administered to the mammal irrespective of consumption ofa potentially antigenic food or protein. In one embodiment, thepotentially antigenic protein is gluten. In one embodiment, thepotentially antigenic protein is one or more wheat proteins.

In one embodiment, intestinal inflammation is characterized byinfiltration and/or proliferation of IELs in the intestine. Accordingly,in one aspect, this invention is directed to a method for attenuating orpreventing intestinal inflammation due to the presence of peptidic foodantigen(s) in the intestine of a mammal, which method comprisesadministering to the mammal an effective amount of a pharmaceuticalcomposition comprising at least one Nepenthes enzyme. In one embodiment,the at least one Nepenthes enzyme is nepenthesin I, nepenthesin II,neprosin, a variant thereof, or a mixture thereof. In one embodiment,the amount of the pharmaceutical composition is effective to attenuateor prevent intestinal inflammation due to the presence of the peptidicfood antigen(s). In one embodiment, the intestinal inflammation is dueto incomplete digestion of a potentially antigenic food protein byendogenous gastric and/or intestinal enzymes. In one embodiment, thecomposition is administered to the mammal prior to ingestion of apotentially antigenic food or protein. In one embodiment, thecomposition is administered to the mammal with ingestion of apotentially antigenic food or protein. In one embodiment, thecomposition is administered to the mammal after ingestion of apotentially antigenic food or protein. In one embodiment, thecomposition is administered to the mammal irrespective of consumption ofa potentially antigenic food or protein. In one embodiment, thepotentially antigenic protein is gluten. In one embodiment, thepotentially antigenic protein is one or more wheat proteins.

In one aspect, this invention is directed to a method for attenuating orpreventing intraepithelial lymphocytosis due to the presence of peptidicfood antigen(s) in an intestine of a mammal, which method comprisesadministering to the mammal an effective amount of a pharmaceuticalcomposition comprising at least one Nepenthes enzyme. In one embodiment,the at least one Nepenthes enzyme is nepenthesin I, nepenthesin II,neprosin, a variant thereof, or a mixture thereof. In one embodiment,the amount of the pharmaceutical composition is effective to inhibitintraepithelial lymphocytosis in the intestine. In one embodiment, thecomposition is administered to the mammal prior to ingestion of apotentially antigenic food or protein. In one embodiment, thecomposition is administered to the mammal with ingestion of apotentially antigenic food or protein. In one embodiment, thecomposition is administered to the mammal after ingestion of apotentially antigenic food. In one embodiment, the composition isadministered to the mammal irrespective of consumption of a potentiallyantigenic food or protein. In one embodiment, the potentially antigenicprotein is gluten. In one embodiment, the potentially antigenic proteinis one or more wheat proteins.

In one embodiment, the effective amount of the pharmaceuticalcomposition is between about 1 mg and about 1 g. In one embodiment, theeffective amount of the pharmaceutical composition depends on the amountof potentially antigenic protein consumed.

In one embodiment, this invention is directed to treating and/orameliorating at least one symptom associated with an immune response tothe presence of gluten or other antigenic protein in the intestine of apatient. Symptoms include, without limitation, “foggy mind”, depression,anxiety, ADHD-like behavior, abdominal pain, bloating, diarrhea,constipation, headaches, migraines, bone or joint pain, chronic fatigue,small intestine damage, development of tissue transglutaminase (tTG)antibodies, severe acne, vomiting, weight loss, irritability,iron-deficiency anemia, arthritis, tingling numbness in the extremities,infertility, and canker sores of the mouth.

In one aspect, this invention is directed to a method for attenuating orpreventing villous atrophy due to the presence of peptidic foodantigen(s) in an intestine of a mammal, which method comprisesadministering to the mammal an effective amount of a pharmaceuticalcomposition comprising at least one Nepenthes enzyme. In one embodiment,the at least one Nepenthes enzyme is nepenthesin I, nepenthesin II,neprosin, a variant thereof, or a mixture thereof. In one embodiment,the potentially antigenic protein is degraded by the pharmaceuticalcomposition so as to inhibit villous atrophy in the intestine. In oneembodiment, the potentially antigenic protein is gluten. In oneembodiment, the potentially antigenic protein is one or more wheatproteins.

In one aspect, this invention is directed to a method for reducing Tcell response to a peptidic food antigen, the method comprisingcontacting the peptidic food antigen with an effective amount of apharmaceutical composition comprising at least one Nepenthes enzyme. Inone embodiment, the at least one Nepenthes enzyme is nepenthesin I,nepenthesin II, neprosin, a variant thereof, or a mixture thereof, underconditions wherein said antigen is degraded so as to reduce T cellresponse to the antigen. In one embodiment, T cell response in anintestine of a mammal is reduced. In one embodiment, the antigen iscontacted with the pharmaceutical composition in the stomach of amammal. In one embodiment, the antigen is contacted with thepharmaceutical composition ex vivo. In one embodiment, the antigen isgluten. In one embodiment, the antigen is an immunotoxic gluten protein.

In one aspect, this invention is directed to a method for attenuating orpreventing a manifestation of celiac disease arising from the presenceof partially hydrolyzed wheat protein in an intestine of a patienthaving celiac disease, comprising administering to the patient aneffective amount of a pharmaceutical composition comprising at least oneNepenthes enzyme. In one embodiment, the at least one Nepenthes enzymeis nepenthesin I, nepenthesin II, neprosin, variant thereof, or amixture thereof, so as to attenuate or prevent a manifestation of celiacdisease.

In one aspect, this invention is directed to a method for improvingdigestibility of a protein from a food in a mammal with an intestinaldisorder, which method comprises administering to the mammal aneffective amount of a pharmaceutical composition comprising at least oneNepenthes enzyme. In one embodiment, the at least one Nepenthes enzymeis nepenthesin I, nepenthesin II, neprosin, variant thereof, or amixture thereof, under conditions wherein the protein in the food isdegraded by the pharmaceutical composition. In one embodiment,degradation of the protein improves absorption of the protein in theintestine. In one embodiment, at least one symptom of the disorder isattenuated or prevented. In one embodiment, the intestinal disorder isCrohn's disease, irritable bowel syndrome, or colitis. In oneembodiment, protein absorption from the food is increased.

In one aspect, this invention is directed to a method for treatinginsufficiency of pancreatic enzymes in a patient in need thereof,comprising administering to the patient an effective amount of apharmaceutical composition comprising at least one Nepenthes enzyme. Inone embodiment, the at least one Nepenthes enzyme is nepenthesin I,nepenthesin II, neprosin, variant thereof, and a mixture thereof. In oneembodiment, one or more pancreatic enzymes in administered. The one ormore pancreatic enzymes may be administered concurrently with thepharmaceutical composition, or at a different time. In one embodiment,the pancreatic enzyme is a lipase, an amylase, a protease, or a mixturethereof. In one embodiment, the insufficiency of pancreatic enzymes isdue to pancreatitis, cystic fibrosis, Shwachman-Bodian-Diamond syndrome,gallstones, lupus, celiac sprue, pancreatic cancer, or pancreaticsurgery. In one embodiment, the pancreatitis is chronic pancreatitis.

In one embodiment, the Nepenthes enzyme is concentrated, isolated, orextracted from the pitcher fluid of a Nepenthes plant. In oneembodiment, the Nepenthes enzyme comprises recombinant nepenthesin I,recombinant nepenthesin II, recombinant neprosin, a variant thereof, ora mixture thereof.

In one embodiment, the variant thereof comprises a protein, the aminoacid sequence of which has at least 85% sequence homology to the aminoacid sequence selected from the group consisting of SEQ ID NO.:1, SEQ IDNO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQID NO.: 20, and SEQ ID NO.: 21. In one embodiment, the variant thereofcomprises a protein, the amino acid sequence of which has at least 85%sequence homology to the amino acid encoded by the cDNA selected fromthe group consisting of SEQ ID NO.:2, SEQ ID NO.:4, and SEQ ID NO.:14.

In one embodiment, the food is a liquid. In one aspect, the food is asolid. In a preferred embodiment, the pharmaceutical composition isorally administered.

Even when a patient adheres to a strict gluten-free diet, gluten is hardto avoid. Numerous foods, particularly processed foods, are contaminatedwith small amounts of gluten. Consumption of even minute amounts ofgluten can lead to a recurrence of symptoms in a patient with celiacdisease. Such is also true of other potentially immunogenic foods.

In one embodiment, the pharmaceutical composition is administeredirrespective of whether the patient has ingested (e.g., knowinglyingested) a food containing a potentially immunogenic protein. In oneembodiment, the pharmaceutical composition is administered on anas-needed basis, e.g., before, during, and/or after a meal that might becontaminated by a potentially immunogenic protein, or in which thepotentially immunogenic protein content is unknown. In one embodiment,the pharmaceutical composition is administered on a regular basis. Inone embodiment, the pharmaceutical composition is administered at leastone time per day. In one embodiment, the pharmaceutical composition isadministered two, three, four, or more times per day. In one embodiment,the pharmaceutical composition is administered in conjunction with(e.g., before, during, or after) each meal and/or snack. In oneembodiment, the pharmaceutical composition is included as part of asustained release formulation where there is a continuous release ofenzyme(s) to allow for intermittent snacking, etc. without regard to theantigenic protein content of the food.

In one embodiment, the pharmaceutical composition is maintained in anaqueous system at about pH 2 wherein the free amino groups of saidenzyme are charged. In one embodiment, the composition is maintained atneutral pH prior to contact with acids in the stomach. In oneembodiment, the pharmaceutical composition comprises a pharmaceuticallyacceptable buffer, such that the pH of the composition remains at pH 5or 6 upon contact with acids in the stomach.

In one embodiment, the effective amount of pharmaceutical composition isbetween about 1 mg and about 1 g. In one embodiment, the effectiveamount of pharmaceutical composition is between about 1 mg and about 1 gper 1 g substrate (e.g., gluten or other potentially immunogenicprotein). In one embodiment, the pharmaceutical composition comprisesmore than one of nepenthesin I, nepenthesin II, neprosin, or a variantthereof.

In one embodiment, the mammal is a human. In one aspect, the humansuffers from gluten sensitivity or celiac disease. In one aspect, it iscontemplated that intestinal antigen protein sensitivity correlates,directly or indirectly, with attention deficit hyperactivity disorder,autism, rheumatoid arthritis, fibromyalgia, and/or dermatitisherpetiformis. It is further contemplated that removing such antigenicintestinal proteins from the intestine using compositions of thisinvention will have a positive effect on attention deficit hyperactivitydisorder, autism, rheumatoid arthritis, fibromyalgia, and/or dermatitisherpetiformis. In a preferred embodiment, the human suffers from celiacdisease.

In one aspect, this invention is directed to a pharmaceuticalcomposition comprising nepenthesin I, nepenthesin II, neprosin, variantthereof, or a mixture thereof. In a preferred embodiment, thepharmaceutical composition comprises neprosin or a variant and/or saltthereof. In a further preferred embodiment, the pharmaceuticalcomposition further comprises at least one additional Nepenthes enzyme.In one embodiment, the additional Nepenthes enzyme comprises nepenthesinI, nepenthesin II, a variant thereof, and/or a salt thereof.

Without being bound by theory, it is believed that nepenthesin I,nepenthesin II, and neprosin are less active or substantially inactiveat neutral to basic pH. This can be important where there is a potentialfor undesirable digestion by the enzyme(s). For example, where thepharmaceutical composition is administered orally, buffering of thecomposition to pH 6.5 or greater may result in a less active form of theenzyme(s) such that the oral mucosa, esophageal mucosa, and other cellsthat may come into contact with the composition will not be digested bythe enzyme(s) therein. Likewise, when the composition is added to afood, the buffered enzyme(s) will be unable to (or less able to) digestthe food before it is consumed. In such situations, introduction of thecomposition to the acidic environment of the stomach will result in adecrease in the pH and activation of enzyme(s).

In one embodiment, the pharmaceutical composition is buffered to aboutpH 6.5 or higher. In a preferred embodiment, the composition is bufferedto about pH 6.5 to about pH 8.5. In one embodiment, the composition isin liquid form. In one embodiment, the composition is in solid form. Inone embodiment, the pH of the composition is adjusted in liquid form andthe composition is dried to form a solid.

In one embodiment, the pharmaceutical composition comprises one or moreadditional proteases. In one embodiment, the one or more additionalprotease is an aspartic protease, a serine protease, a threonineprotease, a cysteine protease, a glutamic acid protease, or ametalloprotease. In one embodiment, the pharmaceutical compositioncomprises one or more additional exoproteases, such as, leucineaminopeptidases and carboxypeptidases. In one embodiment, the one ormore additional protease is trypsin. In a preferred embodiment, the oneor more additional protease is active at acidic pH (e.g., pH 2-6).

In one aspect, the invention is directed to a formulation comprising thepharmaceutical composition of the invention, wherein the enzyme(s) ispresent in a delayed release vehicle such that the enzyme(s) is releasedcontinuously while the formulation is present in the stomach. In oneembodiment, the formulation has a pH of greater than about 5 prior tocontact with acids in the stomach. In one embodiment, the formulationcomprises a biologically acceptable buffer, such that the pH of thecomposition remains at about pH 5 or 6 for at least a period of timeupon contact with acids in the stomach.

In one embodiment, the invention is directed to a unit dose formulationof the pharmaceutical composition. For example and without limitation,the unit dose may be present in a tablet, a capsule, and the like. Theunit dose may be in solid, liquid, powder, or any other form. Withoutbeing bound by theory, it is envisioned that a unit dose formulation ofthe pharmaceutical composition will allow for proper dosing (e.g., basedon the amount of immunogenic protein ingested) while avoiding potentialnegative side effects of administering an excessive amount of thecomposition.

In one embodiment, the invention is directed to a proenzyme form of thenepenthesin I, nepenthesin II, neprosin, and/or variant thereof. In oneembodiment, a propeptide is present on the enzyme. In a preferredembodiment, the propeptide is removed by acidic pH, thereby activatingthe enzyme. In one embodiment, the propeptide comprises thenaturally-occurring propeptide amino acid sequence for the enzyme. Inone embodiment, the propeptide is an artificial propeptide or ameterologous propeptide (i.e., an acid-labile propeptide from adifferent protein and/or species).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an alignment of the protein sequences for nepenthesin Ifrom Nepenthes mirabilis (SEQ ID NO.: 5), Nepenthes alata (SEQ ID NO.:6), Nepenthes gracilis (SEQ ID NO.: 7), Zea mays (SEQ ID NO.: 10), andOryza sativa (SEQ ID NO.: 11), and nepenthesin II from Nepenthesmirabilis (SEQ ID NO.: 8), Nepenthes gracilis (SEQ ID NO.: 9), Oryzasativa (SEQ ID NO.: 12), and Zea mays (SEQ ID NO.: 13).

FIG. 2 indicates the sizes of recombinant nepenthesin proteins. A:Coomassie-stained gel of nepenthesin I. B: MALDI-TOF MS analysis of acidactivated nepenthesin I. C: Coomassie-stained gel of nepenthesin II. D:MALDI-TOF MS analysis of acid activated nepenthesin II.

FIG. 3 indicates the sizes of natural nepenthesin I and nepenthesin II(pooled from 2-3 species) by MALDI-TOF MS.

FIG. 4 is a photograph of a Coomassie-stained gel SDS-PAGE gelindicating the molecular weights of gluten fragments after digestionwith recombinant nepenthesin II, Nepenthes extract, or pepsin.

FIG. 5A is a photograph of vials containing a slurry of gluten proteindigested with pepsin (40 μg) or the indicated amount of recombinantnepenthesin I or recombinant nepenthesin II. FIG. 5B is a photograph ofvials containing a slurry of gluten protein digested with pepsin (40 μg)or the indicated amount of Nepenthes extract. The vials incubated withnepenthesin or Nepenthes extract are less cloudy than the pepsin vial,showing more vigorous digestion of gluten.

FIG. 6 shows the average length of all peptides identified fromdigestion of gliadin from wheat with enriched Nepenthes fluid, usingLC-MS/MS, after 1, 5, 10, 15, 30, 60, 130, 360 or 810 minutes at 37° C.A 95% confidence cut-off (p<0.05) on the scores were used to REDUCEfalse positive identification. Relative standard deviation of thepeptide length is shown in the inset figure.

FIG. 7 displays the number of peptides identified by LC-MS/MS after 1,5, 10, 15, 30, 60, 130, 360 or 810 minutes digestion at 37° C., groupedby length. Data as in FIG. 6.

FIG. 8 displays the same data as in FIG. 6, as a cumulative probabilityof obtaining a certain length after 10, 60, 120, 360 or 810 minutesdigestion at 37° C.

FIG. 9 shows cleavage preferences at (A) the P1 or N-terminal side ofthe cleavage site and at (B) the P1′ or C-terminal side of the cleavagesite for the indicated enzymes. Left bars for each residue indicatedigestion with Nepenthes extract, the middle bars indicate digestionwith purified Nepenthes extract, and the right bars with recombinantnepenthesin I. The % cleavage represents the number of observedcleavages at the given residue, relative to the total number of peptidespresent. Data were obtained from digests of gliadin.

FIG. 10 shows the ion exchange purification profile for Nepenthes fluid.Peaks corresponding to neprosin and nepenthesin are indicated by arrows.The boxed region indicates the collected fractions.

FIG. 11 shows body weights of mice during the course of treatment.Negative control () animals were not challenged with gliadin. Positivecontrol (▪) animals were challenged with gliadin digested by pepsin.Treatment 1 (▴) animals were challenged with gliadin digested withNepenthes extract. Treatment 2 (▾) animals were challenged with gliadindigested with recombinant nepenthesin II.

FIG. 12 is a photograph of the immunohistochemistry for CD3-positiveIELs in the intestine of treated mice.

FIG. 13 shows the average number of CD3-positive intraepitheliallymphocytes (IELs) per 100 enterocytes in the intestine for eachtreatment group. *p<0.05; ***p<0.001

FIG. 14 shows the average villous to crypt ratios for each treatmentgroup.

FIG. 15A shows a sampling of the portions of gliadin that are digestedby neprosin, as detected by data-dependent LC-MS/MS.

FIG. 15B shows the digestion profile of gliadin after digestion withneprosin. Periods indicate cleavage sites.

FIG. 16 shows the location of polymorphisms in the amino acid sequenceof neprosin from different species of Nepenthes.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It is to be understood that this invention is not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of this invention will be limited only by theappended claims.

The detailed description of the invention is divided into varioussections only for the reader's convenience and disclosure found in anysection may be combined with that in another section.

I. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All technical and patentpublications cited herein are incorporated herein by reference in theirentirety. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers. “Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. For example, a composition consistingessentially of the elements as defined herein would not exclude otherelements that do not materially affect the basic and novelcharacteristic(s) of the claimed invention. “Consisting of” shall meanexcluding more than trace amount of other ingredients and substantialmethod steps recited. Embodiments defined by each of these transitionterms are within the scope of this invention.

As used herein, a “potentially antigenic food or protein” is any food orprotein that can cause an immune and/or inflammatory response in theintestine of a sensitive individual. In a preferred embodiment, theindividual is a human and the food is a food intended for humanconsumption. Potentially antigenic foods include, without limitation,wheat, rye, barley, peanuts, nuts and seeds. In one embodiment,potentially antigenic proteins from these foods include prolaminproteins, 2S albumins, non-specific lipid transfer proteins,bifunctional α-amylase/protease inhibitors, soybean hydrophobic protein,indolines, gluten, serpins, purinins, alpha-amylase/protease inhibitors,globulins, and farinins. In a preferred embodiment, the potentiallyantigenic protein (or peptide) is rich in proline and/or glutamineresidues. In an especially preferred embodiment, the potentiallyantigenic protein is gluten. In another preferred embodiment, thepotentially antigenic protein is a wheat protein.

As used herein, the term “gluten” generally refers to the proteinspresent in wheat or related grain species, including barley and rye,which have potential harmful effect to certain individuals. Glutenproteins include gliadins such as α-gliadins, β-gliadins, γ-gliadins andω-gliadins, which are monomeric proteins, and glutenins, which arehighly heterogeneous mixtures of aggregates of high-molecular-weight andlow-molecular-weight subunits held together by disulfide bonds. Manywheat gluten proteins have been characterized. See, for example, Woychiket al., Amino Acid Composition of Proteins in Wheat Gluten, J. Agric.Food Chem., 9(4), 307-310 (1961). The term gluten as used herein alsoincludes oligopeptides that can be derived from normal human digestionof gluten proteins from gluten containing foods and cause the abnormalimmune response. Some of these oligopeptides are resistant to normaldigestive enzymes. Gluten, including the above-mentioned proteins andoligopeptides, is believed to act as an antigen for T cells (e.g., IELs)in patients with gluten intolerance (e.g., celiac sprue). The termgluten also refers to denatured gluten, such as would be found in bakedproducts.

As used herein, the term “gluten sensitivity and related conditions”refers to any condition stemming from intolerance or sensitivity togluten proteins or peptides. These include, without limitation, celiacsprue (celiac disease), wheat allergy, gluten sensitivity,gluten-sensitive enteropathy, idiopathic gluten sensitivity, anddermatitis herpetiformis. Related conditions also include, withoutlimitation, autism, attention deficit hyperactivity disorder (AMID),rheumatoid arthritis, fibromyalgia, Crohn's disease, nutrientmaladsorption, and irritable bowel syndrome (IBS).

The term “neprosin” refers to a prolyl endoprotease with a molecularweight of approximately 29 kilo Daltons (kDa). Neprosin can be isolatedfrom the pitcher secretions of Nepenthes species. Neprosin cleavesproteins carboxy-terminal to proline, with high specificity. The enzymeis active at about pH 2 to about pH 6. In one embodiment, neprosin hasthe amino acid sequence of SEQ ID NO.: 1. The neprosin amino acidsequence is not homologous to any other known protein. In oneembodiment, neprosin is encoded by the cDNA sequence of SEQ ID NO.: 2.In one embodiment, neprosin comprises a signal sequence. In oneembodiment, the signal sequence comprises the amino acid sequence of SEQID NO.: 3. In one embodiment, neprosin does not comprise a signalsequence.

Neprosin includes all isoforms, isotypes, and variants of neprosin,recombinant neprosin, and salts thereof. Salts refer to those saltsformed by neprosin with one or more base or one or more acid whichretain the biological effectiveness and properties of the free neprosin,and which are not biologically or otherwise undesirable. Salts derivedfrom inorganic bases include, but are not limited to, the sodium,potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,manganese, aluminum salts and the like. Salts derived from organic basesinclude, but are not limited to, salts of primary, secondary, andtertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines and basic ion exchange resins, such asisopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine,caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine,glucosamine, methylglucamine, theobromine, purines, piperazine,piperidine, N-ethylpiperidine, polyamine resins and the like. Acids thatcan form salts include, but are not limited to, inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid,malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicyclic acid and thelike.

Examples of proteases include, without limitation, aspartic proteases,serine proteases, threonine proteases, cysteine proteases, glutamic acidproteases, and metalloproteases. Proteases that can be useful in thepresent invention include, without limitation, BACE, cathepsin D,cathepsin E, chymosin (or “rennin”), napsin, pepsin, plasmepsin,presenilin, renin, trypsin, chemotrypsin, elastase, and cysteineendoprotease (EP) B2 (also known as EPB2). Proteases include thosedescribed, for example, in U.S. Pat. Nos. 7,320,788; 7,303,871;7,320,788; 7,628,985; 7,910,541; and 7,943,312; PCT Pat. Pub. Nos.2005/107786; 2008/115428; 2008/115411; 2010/021752; 2010/042203;2011/097266 each of which is expressly incorporated herein by reference.In a preferred embodiment, the at least one additional protease isactive at acidic pH, such as that found in the stomach (e.g., pH 1.5 to3.5).

The term “nepenthesin” refers to the aspartic protease having the EnzymeCommission number EC 3.4.23.12, and includes all isoforms, isotypes, andvariants of nepenthesin such as nepenthesin I and nepenthesin II,nepenthesin isoforms, and recombinant nepenthesin, and salts thereof.Nepenthesin (EC 3.4.23.12) is an aspartic protease of plant origin thatcan be isolated or concentrated from a variety of plant sources, such asthe pitcher secretions of Nepenthes, a carnivorous pitcher plant,commonly known as monkey cups in tropical regions. Nepenthesin isdescribed in detail in U.S. patent application Ser. No. 13/843,369,filed Mar. 15, 2013, which is incorporated herein by reference in itsentirety. Sequence alignment of the known nepenthesin protein sequences(and putative nepenthesin protein sequences) is shown in FIG. 1.

In one embodiment, “effective amount” refers to that amount of acomposition that results in inhibition or amelioration of symptoms in asubject or a desired biological outcome, e.g., improved clinical signs,delayed onset of disease, etc. The effective amount can be determined byone of ordinary skill in the art. The selected dosage level can dependupon the severity of the condition being treated, and the condition andprior medical history of the mammal being treated. However, it is withinthe skill of the art to start doses of the composition at levels lowerthan required to achieve the desired therapeutic effect and to graduallyincrease the dosage until the desired effect is achieved.

The term “manifestations of celiac disease” refers to any of thesymptoms or clinical presentations of celiac disease. Suchmanifestations include, without limitation, intestinal inflammation,“foggy mind”, depression, anxiety, ADHD-like behavior, abdominal pain,bloating, diarrhea, constipation, headaches, migraines, bone or jointpain, chronic fatigue, small intestine damage, development of tissuetransglutaminase (tTG) antibodies, severe acne, vomiting, weight loss,irritability, iron-deficiency anemia, arthritis, tingling numbness inthe extremities, infertility, and canker sores of the mouth.Manifestations further include small intestinal mucosal villous atrophywith crypt hyperplasia, mucosal inflammation of the intestine,malabsorption of nutrients, abdominal distension, as well as asubstantially enhanced risk for the development of osteoporosis andintestinal malignancies (lymphoma and carcinoma).

“Concurrent administration,” or “co-treatment,” as used herein includesadministration of the agents together, or before or after each other.

The term “modulate,” “attenuate” or “ameliorate” means any treatment ofa disease or disorder in a subject, such as a mammal, including:

-   -   preventing or protecting against the disease or disorder, that        is, causing the abnormal biological reaction or symptoms not to        develop;    -   inhibiting the disease or disorder, that is, arresting or        suppressing the development of abnormal biological reactions        and/or clinical symptoms; and/or    -   relieving the disease or disorder, that is, causing the        regression of abnormal biological reactions and/or symptoms.

As used herein, the term “preventing” or “inhibiting” refers to theprophylactic treatment of a subject in need thereof. The prophylactictreatment can be accomplished by providing an appropriate dose of atherapeutic agent to a subject at risk of suffering from an ailment,thereby substantially averting onset of the ailment.

As used herein, the term “condition” refers to a disease state for whichthe compounds, compositions and methods provided herein are being used.

As used herein, the term “patient” or “subject” refers to mammals andincludes humans and non-human mammals. In particular embodiments herein,the patient or subject is a human.

The term “about” when used before a numerical value indicates that thevalue may vary within a reasonable range: ±5%, ±1%, or ±0.2%.

A polynucleotide or polynucleotide region (or a polypeptide orpolypeptide region) having a certain percentage (for example, 80%, 85%,90%, or 95%) of “sequence identity” to another sequence means that, whenaligned, that percentage of bases (or amino acids) are the same incomparing the two sequences. The alignment and the percent homology orsequence identity can be determined using software programs known in theart, for example those described in Current Protocols in MolecularBiology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table7.7.1. Preferably, default pardmeters are used for alignment. Onealignment program is BLAST, using default parameters. Examples of theprograms include BLASTN and BLASTP, using the following defaultparameters: Genetic code=standard; filter=none; strand=both; cutoff=60;expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGHSCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR. Details of these programs can befound at the following Internet address: ncbi.nlm.nih.gov/cgi-bin/BLAST.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare homologous at that position. A-degree of homology between sequencesis a function of the number of matching or homologous positions sharedby the sequences. An “unrelated” or “non-homologous” sequence sharesless than 40% identity, or alternatively less than 25% identity, withone of the sequences of the present disclosure.

II. METHODS

In one aspect, this invention relates to methods for modulating acondition mediated by gluten intolerance in a patient, comprisingadministering to the patient an effective amount of a pharmaceuticalcomposition comprising a Nepenthes enzyme. In a preferred embodiment,the condition is celiac disease or a wheat allergy.

In another aspect, this invention relates to a method for attenuating orpreventing production and/or recruitment of IELs in the intestine due tothe presence of a peptidic food antigen in an intestine of a mammal. Inone embodiment, the method comprises administering to the mammal aneffective amount of a pharmaceutical composition comprising a Nepenthesenzyme. In one embodiment, the gluten protein is degraded by thepharmaceutical composition so as to attenuate or prevent productionand/or recruitment of IELs in the intestine.

In one aspect, this invention relates to a method for attenuating orpreventing intestinal inflammation due to the presence of a peptidicfood antigen in the intestine of a mammal. In one embodiment, the methodcomprises administering to the mammal an effective amount of apharmaceutical composition comprising a Nepenthes enzyme. In oneembodiment, the peptidic food antigen is degraded by the enzyme(s) so asto attenuate or prevent intestinal inflammation.

In one aspect, this invention relates to a method for attenuating orpreventing intraepithelial lymphocytosis due to the presence of apeptidic food antigen in an intestine of a mammal. In one embodiment,the method comprises administering to the mammal an effective amount ofa pharmaceutical composition comprising a Nepenthes enzyme. In oneembodiment, the peptidic food antigen is degraded by the pharmaceuticalcomposition so as to attenuate or prevent intraepithelial lymphocytosisin the intestine.

In one aspect, this invention relates to a method for attenuating orpreventing villous atrophy due to the presence of a peptidic foodantigen in an intestine of a mammal. In one embodiment, the methodcomprises administering to the mammal an effective amount of apharmaceutical composition comprising a Nepenthes enzyme. In oneembodiment, the peptidic food antigen is degraded by the pharmaceuticalcomposition so as to attenuate or prevent villous atrophy in theintestine. In one embodiment, the villous atrophy is a result ofinflammation of the intestine.

In one embodiment, the Nepenthes enzyme is nepenthesin I, nepenthesinII, neprosin, variant thereof, or a mixture thereof. In a preferredembodiment, the pharmaceutical formulation is a sustained releaseformulation.

In one embodiment, the variant is a protein having an amino acidsequence having at least 85% sequence homology to the amino acidsequence of SEQ ID NO.: 1, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7,SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.:20, or SEQ ID NO.:21. In oneembodiment, the variant is a protein having an amino acid sequencehaving at least 85% sequence homology to the amino acid sequence of SEQID NO.: 1. In one embodiment, the variant is a protein having an aminoacid sequence having at least 85% sequence homology to the amino acidsequence of SEQ ID NO.: 5. In one embodiment, the variant is a proteinhaving an amino acid sequence having at least 85% sequence homology tothe amino acid sequence of SEQ ID NO.: 6. In one embodiment, the variantis a protein having an amino acid sequence having at least 85% sequencehomology to the amino acid sequence of SEQ ID NO.: 7. In one embodiment,the variant is a protein having an amino acid sequence having at least85% sequence homology to the amino acid sequence of SEQ ID NO.: 8. Inone embodiment, the variant is a protein having an amino acid sequencehaving at least 85% sequence homology to the amino acid sequence of SEQID NO.: 9. In one embodiment, the variant is a protein having an aminoacid sequence having at least 85% sequence homology to the amino acidsequence of SEQ ID NO.: 20. In one embodiment, the variant is a proteinhaving an amino acid sequence having at least 85% sequence homology tothe amino acid sequence of SEQ ID NO.: 21.

In one embodiment, the pharmaceutical composition comprises an extractof Nepenthes pitcher fluid. In one embodiment, the pharmaceuticalcomposition comprises nepenthesin I, nepenthesin II, and/or neprosinpurified from an extract of Nepenthes pitcher fluid. In one embodiment,at least one of nepenthesin I, nepenthesin II, neprosin, or variantthereof is a recombinant protein. In one embodiment, the pharmaceuticalcomposition is between about pH 5 and about pH 8 prior toadministration. Pharmaceutical compositions for use in the methodsdescribed herein are discussed in more detail below.

In a preferred embodiment, the mammal is a human. In one embodiment, thehuman suffers from a disease selected from the group consisting ofgluten intolerance, celiac disease, attention deficit hyperactivitydisorder, autism, rheumatoid arthritis, fibromyalgia, and dermatitisherpetiformis. In one embodiment, the human suffers from a food allergy.

In one embodiment, the pharmaceutical composition is orally administeredprior to, during, or immediately after consumption of agluten-containing food.

In some embodiments, the pharmaceutical composition is administered tothe subject prior to ingestion by the subject of the food comprisinggluten or suspect of comprising gluten. In some embodiments, thepharmaceutical composition is administered within a period that theenzyme is at least partially effective (for example, at least about 10%,20%, 50%, 70%, 90% of original activity) in degrading gluten in the foodthat the subject will ingest. In some embodiments, the pharmaceuticalcomposition is administered not more than about 4 hours, 3 hours, 2hours, 1 hour, or 30 minutes prior to ingestion of the food by thesubject.

In some embodiments, the pharmaceutical composition is administered tothe subject concurrently with ingestion by the subject of thepotentially immunogenic food. In some embodiments, the enzymecomposition is administered with the food. In some embodiments, thepharmaceutical composition is administered separately from the food.

In some embodiments, the pharmaceutical composition is administered tothe subject shortly after ingestion by the subject of the potentiallyimmunogenic food. In some embodiments, the pharmaceutical composition isadministered within a period that at least part (for example, at leastabout 10%, 20%, 50%, 70%, 90%) of the antigen(s) in the food is still inthe stomach of the subject. In some embodiments, the pharmaceuticalcomposition is administered not more than 4 hours, 3 hours, 2 hours, 1hour, or 30 minutes after ingestion of the food by the subject.

Typically, the pharmaceutical composition is administered in an amountthat is safe and sufficient to produce the desired effect ofdetoxification of peptidic food antigen(s). The dosage of thepharmaceutical composition can vary depending on many factors such asthe particular enzyme administered, the subject's sensitivity to thefood, the amount and types of antigen-containing food ingested, thepharmacodynamic properties of the enzyme, the mode of administration,the age, health and weight of the recipient, the nature and extent ofthe symptoms, the frequency of the treatment and the type of concurrenttreatment, if any, and the clearance rate of the enzyme. One of skill inthe art can determine the appropriate dosage based on the above factors.The composition may be administered initially in a suitable dosage thatmay be adjusted as required, depending on the clinical response. Invitro assays may optionally be employed to help identify optimal dosageranges. The precise dose to be employed in the formulation will alsodepend on the route of administration and/or the seriousness of thedisease or disorder, and should be decided according to the judgment ofthe practitioner and each subject's circumstances.

The dosage or dosing regimen of an adult subject may be proportionallyadjusted for children and infants, and also adjusted for otheradministration or other formats, in proportion for example to molecularweight or immune response. Administration or treatments may be repeatedat appropriate intervals, at the discretion of the physician.

Generally, the pharmaceutical composition is administered when needed,such as when the subject will be or is consuming or has consumed a foodcomprising an antigenic protein or suspected of comprising an antigenicprotein. In any case, it can be administered in dosages of about 0.001mg to about 1000 mg of enzyme per kg body weight per day, or about 1 mgto about 100 g per dose for an average person. In some embodiments, theenzyme can be administered at 0.001, 0.01, 0.1, 1, 5, 10, 50, 100, 500,or 1000 mg/kg body weight per day, and ranges between any two of thesevalues (including endpoints). In some embodiments, the enzyme can beadministered at 1 mg, 10 mg, 100 mg, 200 mg, 500 mg, 700 mg, 1 g, 10 g,20 g, 50 g, 70 g, 100 g per dose, and ranges between any two of thesevalues (including endpoints). In some embodiments, it may beadministered once, twice, three times, etc. a day, depending on thenumber of times the subject ingests a food comprising an antigenicprotein and/or how much of such food is consumed. The amount of enzymerecited herein may relate to total enzyme or each enzyme in thecomposition.

In some embodiments, the amount of pharmaceutical compositionadministered is dependent on the amount (or approximate amount) ofsubstrate (e.g., gluten and/or other protein or potentially antigenicprotein) consumed/to be consumed. In one embodiment, about 1 mg to about1 g of enzyme is administered per 1 g of substrate. In one embodiment,about 5 mg to about 1 g of enzyme is administered per 1 g of substrate.In one embodiment, about 10 mg to about 1 g of enzyme is administeredper 1 g of substrate. In one embodiment, about 100 mg to about 1 g ofenzyme is administered per 1 g of substrate. In one embodiment, about 1mg to about 500 mg of enzyme is administered per 1 g of substrate. Inone embodiment, about 1 mg to about 250 mg of enzyme is administered per1 g of substrate. In one embodiment, about 1 mg to about 100 mg ofenzyme is administered per 1 g of substrate. In one embodiment, about 1mg to about 10 mg of enzyme is administered per 1 g of substrate. Thisincludes any values with these ranges (including endpoints), andsubranges between any two of these values.

In one embodiment, the ratio of substrate to enzyme administered isbetween about 1:1 and about 10000:1. In a preferred embodiment, theratio of substrate to enzyme is between about 10:1 and about 1000:1. Inone embodiment, the ratio of substrate to enzyme is between about 10:1and about 100:1.

The pharmaceutical composition of this invention can be administered asthe sole active agent or they can be administered in combination withother agents (simultaneously, sequentially or separately, or throughco-formulation), including other compounds that demonstrate the same ora similar therapeutic activity and that are determined to safe andefficacious for such combined administration.

In some embodiments, the pharmaceutical composition is administered withan additional enzyme, such as a gastric protease, an aspartic protease(such as pepsin, pepsinogen or those described by Chen et al., Asparticproteases gene family in rice: Gene structure and expression, predictedprotein features and phylogenetic relation, Gene 442:108-118 (2009)),and enzymes such as another prolyl endopeptidase (PEP), dipeptidylpeptidase IV (DPP IV), and dipeptidyl carboxypeptidase (DCP) or cysteineproteinase B (described in U.S. Pat. No. 7,910,541). In one embodiment,the other enzyme is administered in the form of bacteria that produceand/or secrete the additional enzyme. In one embodiment, the bacteriaare engineered to produce and/or secrete nepenthesin I, nepenthesin II,neprosin, and/or a variant thereof.

In some embodiments, the pharmaceutical composition is administered tothe subject with another agent. Non-limiting examples of agents that canbe administered with the pharmaceutical composition include inhibitorsof tissue transglutaminase, anti-inflammatory agents such as amylases,glucoamylases, endopeptidases, HMG-CoA reductase inhibitors (e.g.,compactin, lovastatin, simvastatin, pravastatin and atorvastatin),leukotriene receptor antagonists (e.g., montelukast and zafirlukast),COX-2 inhibitors (e.g., celecoxib and rofecoxib), p38 MAP kinaseinhibitors (e.g., BIRB-796); mast cell-stabilizing agents such as sodiumchromoglycate (chromolyn), pemirolast, proxicromil, repirinast,doxantrazole, amlexanox nedocromil and probicromil, anti-ulcer agents,anti-allergy agents such as anti-histamine agents (e.g., acrivastine,cetirizine, desloratadine, ebastine, fexofenadine, levocetirizine,loratadine and mizolastine), inhibitors of transglutaminase 2 (TG2),anti-TNFα agents, and antibiotics. In one embodiment, the additionalagent is a probiotic. Probiotics include, without limitation,lactobacillus, yeast, bacillus, or bifidobacterium species and strains.In one embodiment, the other agent is elafin. In one embodiment, theother agent is administered in the form of bacteria that produce and/orsecrete the additional agent.

In some embodiments, the other agent comprises an enzyme (e.g.,protease) that is active in the intestine. Without being limited bytheory, it is believed that such enzymes may act synergistically withthe enzyme(s) of the pharmaceutical composition to further degradeimmunogenic proteins.

Also provided herein is the use of an enzyme composition comprisingnepenthesin I, nepenthesin II, neprosin, a variant thereof, and/or asalt thereof in the manufacture of a medicament for the treatment orprevention of one of the aforementioned conditions and diseases.

III. PHARMACEUTICAL COMPOSITIONS

The pharmaceutical composition can be administered in a variety ofcompositions alone or with appropriate, pharmaceutically acceptablecarriers, excipients, or diluents.

Accordingly, in another aspect, provided herein is a compositioncomprising nepenthesin I, nepenthesin II, neprosin, a variant thereof,and/or a salt thereof. In some embodiments, the composition is apharmaceutical composition. The compositions may be formulated intosolid, semi-solid, or liquid forms, such as tablets, capsules, powders,granules, ointments, solutions, injections, gels, and microspheres.Administration of the composition can be achieved in various ways, forexample, by oral administration.

In some embodiments, the pharmaceutical composition comprises atherapeutically effective amount of nepenthesin I, nepenthesin II,neprosin, variant thereof, or mixture thereof and a pharmaceuticallyacceptable carrier. In a particular embodiment, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers.

Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like. Examples of suitablepharmaceutical carriers are described in “Remington's PharmaceuticalSciences” by E. W. Martin, incorporated in its entirety by referenceherein. Such compositions will contain a therapeutically effectiveamount of the enzyme(s), preferably in purified form, together with asuitable amount of carrier so as to provide the form for properadministration to the subject. The formulation should suit the mode ofadministration.

For oral administration, the pharmaceutical composition can be usedalone or in combination with appropriate additives to make tablets,powders, granules, capsules, syrups, liquids, suspensions, etc. Forexample, solid oral forms of the composition can be prepared withconventional additives, disintegrators, lubricants, diluents, bufferingagents, moistening agents, preservatives and flavoring agents.Non-limiting examples of excipients include lactose, mannitol, cornstarch, potato starch, crystalline cellulose, cellulose derivatives,acacia, corn starch, sodium carboxymethylcellulose, talc, magnesiumstearate, flavors and colors. In some embodiments, the formulationreleases the enzyme(s) in the stomach of the subject so that thepeptidic food antigen(s) can be degraded by the enzyme(s).

The composition can be lyophilized from an aqueous solution optionallyin the presence of appropriate buffers (e.g. phosphate, citrate,histidine, imidazole buffers) and excipients (e.g. cryoprotectants suchas sucrose, lactose, trehalose). Lyophilized cakes can optionally beblended with excipients and made into different forms.

In another aspect, provided are methods for treating gluten intoleranceor an associated condition, such as celiac disease, wheat allergy,gluten sensitivity and dermatitis herpetiformis, in a patient in needthereof, comprising treating a food comprising gluten or suspected ofcomprising gluten with an effective amount of the composition prior toconsumption by the patient. In some embodiments, the food is combinedwith an effective amount of the composition during its preparation. Inone embodiment, the composition is added after any heating steps in thefood preparation. In one embodiment, the composition is added before oneor more heating steps in the food preparation.

Nepenthesin I, nepenthesin II, and neprosin occur as proenzymes inNepenthes prior to activation. That is, the protein includes apropeptide that is cleaved in order to activate the enzyme in thepitcher fluid. In one embodiment, the composition comprises nepenthesinI, nepenthesin II, neprosin, a variant thereof, and/or a salt thereofcomprising a propeptide. In one embodiment, the propeptide is adjacentto the N terminus of the enzyme. In one embodiment, the propeptide isthe naturally-occurring propeptide for the enzyme. In one embodiment,the propeptide is a heterologous propeptide (e.g., from a differentprotein or species, or synthetic). In one embodiment, the propeptide iscleaved by acidic conditions. In one embodiment, the propeptide iscleaved by an enzyme. In one embodiment, the presence of the propeptideresults in delayed activity of the enzyme in the stomach (e.g., due tothe time required to remove the propeptide and produce the matureenzyme). In one embodiment, the propeptide is engineered to be removedmore slowly in order to delay activity of the enzyme in the stomach. Inone embodiment, the propeptide is engineered to be removed more quicklyin order to speed up activity of the enzyme in the stomach.

In a preferred embodiment, the formulation is a controlled releaseformulation. The term “controlled release formulation” includessustained release and time-release formulations. Controlled releaseformulations are well-known in the art. These include excipients thatallow for sustained, periodic, pulse, or delayed release of the drug.Controlled release formulations include, without limitation, embeddingof the drug into a matrix; enteric coatings; micro-encapsulation; gelsand hydrogels; and any other formulation that allows for controlledrelease of a drug.

In some embodiments, the composition is administered as a food additivetogether with a food comprising or suspected of comprising a potentiallyantigenic food protein. In one embodiment, the food comprises or issuspected of comprising gluten, for example bread, pasta, cereal, andthe like, made from wheat, rye and barley, etc. In some embodiments, thecomposition is added as an ingredient in such food. In some embodiments,the composition is dispersed into a food prior to consumption,optionally at a pH where it is inactive, such as a pH of about or above5. In some embodiments, the composition can be made or incorporated intoa powder, a spread, a spray, a sauce, a dip, a whipped cream, etc., thatcan be applied to the food when the food is being consumed by a patient.In some embodiments, the composition can be made into forms that appealto one's appetite, such as candies, chewing gums, dietary supplementchews, syrup, etc. for easy administration. In some embodiments, thecomposition can be mixed with common food items, such as sugar, salt,salad dressing, spices, cheese, butter, margarines, spreads, butter,frying shortenings, mayonnaises, dairy products, nut butters, seedbutters, kernel butters, peanut butter, etc. Preferably, the food itemsor additives comprising the composition do not require heating beforebeing ingested by a patient so that possible loss of activity of theenzyme(s) due to elevated temperature can be minimized.

In one embodiment, the enzyme(s) in the composition is activated uponcontact with acid (i.e., in the stomach).

In another aspect, provided is a food product comprising neprosin,nepenthesin I, nepenthesin II, a variant thereof, or a combinationthereof. In some embodiments, the food product comprises gluten or issuspected of comprising gluten, such as bakery products (e.g., cakes,muffins, donuts, pastries, rolls, and bread), pasta, crackers, tortillachips, cereal etc. made from wheat, rye and barley. In some embodiments,the food product can be consumed with another food product comprisinggluten or suspected of comprising gluten. Non-limiting examples of suchfood include a powder, a spread, a spray, a sauce, a dip, a whippedcream, candies, chewing′gums, syrup, sugar, salt, salad dressing,spices, cheese, butter, margarines, spreads, butter, frying shortenings,mayonnaises, dairy products, nut butters, seed butters, kernel butters,peanut butter, etc.

In some embodiments, the composition comprising neprosin, nepenthesin I,nepenthesin II, a variant thereof, or a combination thereof is admixedwith food, or used to pre-treat foodstuffs containing glutens. Thecomposition present in foods can be enzymatically active to reduce thelevel of gluten in the food prior to or during ingestion.

In one aspect of the invention, a composition comprising neprosin,nepenthesin nepenthesin II, a variant thereof, or a combination thereofis added to food before the food is consumed. In one embodiment, theinvention is directed to a dispenser comprising an inner excipient andan effective amount of the pharmaceutical composition to digest gluten.In one embodiment, the pharmaceutical composition and/or inner excipientare added to food before the food is consumed. In one embodiment, thefood comprises gluten or is suspected to comprise gluten. In oneembodiment, the inner excipient comprises sodium chloride or sodiumiodide, or a mixture thereof. In one embodiment, the pharmaceuticalcomposition and/or inner excipient are in granular form, sized toefficiently dispense from said dispenser.

In some embodiments, the composition (such as pharmaceutical compositionor edible composition) or food product comprises from about 0.1% toabout 99%, from about 0.5% to about 95%, from about 1% to about 95%,from about 5% to about 95%, from about 10% to about 90%, from about 20%to about 80%, from about 25% to about 75% of the enzyme(s). In someembodiments, the amount of enzyme in the composition (such aspharmaceutical composition or edible composition) or food product isabout 0.01%, about 0.1%, about 0.5%, about 1%, about 5%, about 10%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, or about 95% of the total composition or foodproduct, or a range between any two of the values (including endpoints).

In some embodiments, the composition comprises neprosin and nepenthesin,or a variant thereof. In some embodiments, the nepenthesin isnepenthesin I and/or nepenthesin II, or a variant thereof. In someembodiments, the nepenthesin is recombinant nepenthesin I and/orrecombinant nepenthesin II, or a variant thereof. In some embodiments,the nepenthesin is recombinant nepenthesin I and recombinant nepenthesinII, or a variant of each thereof. In some embodiments, the neprosin isrecombinant neprosin, or a variant thereof. In a preferred embodiment,the composition comprises nepenthesin I, nepenthesin II, and/or neprosincomprising the amino acid sequence(s) of nepenthesin I, nepenthesin II,and/or neprosin from a Nepenthes species, or a variant(s) thereof.

Nepenthesin I mRNA/cDNA sequences have been described from severalNepenthes species, for example, Nepenthes mirabilis (GenBank AccessionNo. JX494401), Nepenthes gracilis (GenBank Accession No. AB114914), andNepenthes alata (GenBank Accession No. AB266803). Nepenthesin IImRNA/cDNA sequences have been described from several Nepenthes species,for example, Nepenthes mirabilis (GenBank Accession No. JX494402), andNepenthes gracilis (GenBank Accession No. AB114915).

Nepenthesin I protein sequences have been described from severalNepenthes species, for example, Nepenthes mirabilis (GenBank AccessionNo. AFV26024; SEQ ID NO.: 5), Nepenthes gracilis (GenBank Accession No.BAD07474; SEQ ID NO.: 7), and Nepenthes alata (GenBank Accession No.BAF98915; SEQ ID NO.: 6). Nepenthesin II protein sequences have beendescribed from several Nepenthes species, for example, Nepenthesmirabilis (GenBank Accession No. AFV26025; SEQ ID NO.: 8), and Nepenthesgracilis (GenBank Accession No. BAD07475; SEQ ID NO.: 9). The sequencesare also found in U.S. Patent Application Publication No. 2014/0186330,which is incorporated herein by reference in its entirety.

Each of the sequences represented by the GenBank Accession Nos. providedherein are incorporated herein by reference in their entireties.

In some embodiments, the nepenthesin is a variant of nepenthesin havingat least about 85% sequence homology to an amino acid sequence ofnepenthesin I (e.g., SEQ ID NO.: 5; SEQ ID NO.: 6; SEQ ID NO.: 7; or SEQID NO.: 21). In some embodiments, the variant has at least about 90%sequence homology to an amino acid sequence of nepenthesin I. In someembodiments, the variant has at least about 95% sequence homology to anamino acid sequence of nepenthesin I. In some embodiments, the varianthas at least about 96% sequence homology to an amino acid sequence ofnepenthesin 1. In some embodiments, the variant has at least about 97%sequence homology to an amino acid sequence of nepenthesin I. In someembodiments, the variant has at least about 98% sequence homology to anamino acid sequence of nepenthesin I. In some embodiments, the varianthas at least about 99% sequence homology to an amino acid sequence ofnepenthesin I. In one embodiment, the nepenthesin comprises the aminoacid sequence of SEQ ID NO.: 5; SEQ ID NO.: 6; SEQ ID NO.: 7; or SEQ IDNO.: 21.

In some embodiments, the nepenthesin is a variant of nepenthesin havingat least about 85% sequence homology to an amino acid sequence ofnepenthesin H (e.g., SEQ ID NO.: 8; SEQ ID NO.: 9; or SEQ ID NO.: 22).In some embodiments, the variant has at least about 90% sequencehomology to an amino acid sequence of nepenthesin H. In someembodiments, the variant has at least about 95% sequence homology to anamino acid sequence of nepenthesin II. In some embodiments, the varianthas at least about 96% sequence homology to an amino acid sequence ofnepenthesin II. In some embodiments, the variant has at least about 97%sequence homology to an amino acid sequence of nepenthesin II. In someembodiments, the variant has at least about 98% sequence homology to anamino acid sequence of nepenthesin II. In some embodiments, the varianthas at least about 99% sequence homology to an amino acid sequence ofnepenthesin II. In one embodiment, the nepenthesin comprises the aminoacid sequence of SEQ ID NO.: 8; SEQ ID NO.: 9; or SEQ ID NO.: 22.

In one aspect of the invention, the ratio of neprosin to nepenthesin Iand/or II in the composition is such that the peptidic food antigen iscleaved into sufficiently small and/or innocuous fragments so as toprevent gluten intolerance, celiac disease, wheat allergy, or dermatitisherpetiformis, inflammation, IEL proliferation or recruitment,intraepithelial lymphocytosis, and/or villous atrophy, or any symptomthereof, in an intestine of the subject. In some embodiments, theneprosin:nepenthesin ratio is between about 1:100 to about 100:1.

In some embodiments, the composition comprises a ratio of neprosin tonepenthesin (nepenthesin I and/or II) of at least about 100:1. In someembodiments, the composition comprises a ratio of neprosin tonepenthesin of at least about 90:1. In some embodiments, the compositioncomprises a ratio of neprosin to nepenthesin of at least about 70:1. Insome embodiments, the composition comprises a ratio of neprosin tonepenthesin of at least about 60:1. In some embodiments, the compositioncomprises a ratio of neprosin to nepenthesin of at least about 50:1. Insome embodiments, the composition comprises a ratio of neprosin tonepenthesin of at least about 40:1. In some embodiments, the compositioncomprises a ratio of neprosin to nepenthesin of at least about 30:1. Insome embodiments, the composition comprises a ratio of neprosin tonepenthesin of at least about 20:1. In some embodiments, the compositioncomprises a ratio of neprosin to nepenthesin of at least about 10:1. Insome embodiments, the composition comprises a ratio of neprosin tonepenthesin of at least about 5:1. In some embodiments, the compositioncomprises a ratio of neprosin to nepenthesin of at least about 4:1. Insome embodiments, the composition comprises a ratio of neprosin tonepenthesin of at least about 3:1. In some embodiments, the compositioncomprises a ratio of neprosin to nepenthesin of at least about 2:1. Insome embodiments, the composition comprises a ratio of neprosin tonepenthesin of at least about 1:1. In some embodiments, the compositioncomprises a ratio of neprosin to nepenthesin of at least about 1:2. Insome embodiments, the composition comprises a ratio of neprosin tonepenthesin of at least about 1:3. In some embodiments, the compositioncomprises a ratio of neprosin to nepenthesin of at least about 1:4. Insome embodiments, the composition comprises a ratio of neprosin tonepenthesin of at least about 1:5. In some embodiments, the compositioncomprises a ratio of neprosin to nepenthesin of at least about 1:10. Insome embodiments, the composition comprises a ratio of neprosin tonepenthesin of at least about 1:20. In some embodiments, the compositioncomprises a ratio of neprosin to nepenthesin of at least about 1:30. Insome embodiments, the composition comprises a ratio of neprosin tonepenthesin of at least about 1:40. In some embodiments, the compositioncomprises a ratio of neprosin to nepenthesin of at least about 1:50. Insome embodiments, the composition comprises a ratio of neprosin tonepenthesin of at least about 1:60. In some embodiments, the compositioncomprises a ratio of neprosin to nepenthesin of at least about 1:70. Insome embodiments, the composition comprises a ratio of neprosin tonepenthesin of at least about 1:80. In some embodiments, the compositioncomprises a ratio of neprosin to nepenthesin of at least about 1:90. Insome embodiments, the composition comprises a ratio of neprosin tonepenthesin of at least about 1:100.

In one aspect of the invention, the ratio of nepenthesin I tonepenthesin II in the composition is such that the peptidic food antigenis cleaved into sufficiently small and/or innocuous fragments so as toprevent inflammation, IEL proliferation or recruitment, intraepitheliallymphocytosis, and/or villous atrophy in an intestine of the subject. Insome embodiments, the nepenthesin I:nepenthesin II ratio is betweenabout 1:100 to about 100:1.

In some embodiments, the composition comprises a ratio of nepenthesin Ito nepenthesin II of at least about 100:1. In some embodiments, thecomposition comprises a ratio of nepenthesin I to nepenthesin II of atleast about 90:1. In some embodiments, the composition comprises a ratioof nepenthesin I to nepenthesin II of at least about 70:1. In someembodiments, the composition comprises a ratio of nepenthesin I tonepenthesin II of at least about 60:1. In some embodiments, thecomposition comprises a ratio of nepenthesin I to nepenthesin II of atleast about 50:1. In some embodiments, the composition comprises a ratioof nepenthesin I to nepenthesin II of at least about 40:1. In someembodiments, the composition comprises a ratio of nepenthesin I tonepenthesin II of at least about 30:1. In some embodiments, thecomposition comprises a ratio of nepenthesin I to nepenthesin II of atleast about 20:1. In some embodiments, the composition comprises a ratioof nepenthesin I to nepenthesin II of at least about 10:1. In someembodiments, the composition comprises a ratio of nepenthesin I tonepenthesin II of at least about 5:1. In some embodiments, thecomposition comprises a ratio of nepenthesin I to nepenthesin II of atleast about 4:1. In some embodiments, the composition comprises a ratioof nepenthesin I to nepenthesin II of at least about 3:1. In someembodiments, the composition comprises a ratio of nepenthesin I tonepenthesin II of at least about 2:1. In some embodiments, thecomposition comprises a ratio of nepenthesin I to nepenthesin II of atleast about 1:1. In some embodiments, the composition comprises a ratioof nepenthesin I to nepenthesin II of at least about 1:2. In someembodiments, the composition comprises a ratio of nepenthesin I tonepenthesin II of at least about 1:3. In some embodiments, thecomposition comprises a ratio of nepenthesin I to nepenthesin H of atleast about 1:4. In some embodiments, the composition comprises a ratioof nepenthesin I to nepenthesin II of at least about 1:5. In someembodiments, the composition comprises a ratio of nepenthesin I tonepenthesin II of at least about 1:10. In Some embodiments, thecomposition comprises a ratio of nepenthesin I to nepenthesin II of atleast about 1:20. In some embodiments, the composition comprises a ratioof nepenthesin I to nepenthesin II of at least about 1:30. In someembodiments, the composition comprises a ratio of nepenthesin I tonepenthesin II of at least about 1:40. In some embodiments, thecomposition comprises a ratio of nepenthesin I to nepenthesin H of atleast about 1:50. In some embodiments, the composition comprises a ratioof nepenthesin I to nepenthesin II of at least about 1:60. In someembodiments, the composition comprises a ratio of nepenthesin I tonepenthesin II of at least about 1:70. In some embodiments, thecomposition comprises a ratio of nepenthesin I to nepenthesin II of atleast about 1:80. In some embodiments, the composition comprises a ratioof nepenthesin I to nepenthesin II of at least about 1:90. In someembodiments, the composition comprises a ratio of nepenthesin I tonepenthesin II of at least about 1:100.

IV. METHODS OF PREPARATION

It is contemplated that nepenthesin and/or neprosin can be concentrated(or extracted) or purified by methods known in the art, for example (butnot limited to) filtration or affinity purification based on immobilizedpepstatin, from a natural source, including pitcher secretions of plantssuch as Nepenthes. Classical protein chromatography, such as sizeexclusion chromatography (also known as gel permeation chromatography)and/or chromatofocusing chromatography, may also be used to concentrate(or extract) or purify nepenthesin and/or neprosin. Chromatofocusing maybe used prior to or after size exclusion. Nepenthesin I, nepenthesin II,and neprosin are found in relatively small quantity in natural plantsecretions. Production of nepenthesin I, nepenthesin II, and/or neprosincan be increased, for example, using bioengineering technologies tocreate transgenic plants that express and/or secrete increased amountsof the desired enzyme(s), or a variant thereof.

Besides being isolated from a plant source, the Nepenthes enzyme orvariant thereof may be prepared by chemical synthesis. Chemicalsynthesis can be achieved by coupling of the amino acids according tothe sequence of the desired enzyme or variant. Various peptide couplingmethods and commercial peptide synthetic apparatuses are available tosynthesis peptide or proteins, for example, automated synthesizers byApplied Biosystems, Inc., Foster City, Calif., Beckman, and othermanufacturers.

In another aspect, provided is a method of preparing Nepenthes enzyme orvariant thereof using recombinant production systems by transforming ortransfecting a cell with the DNA (e.g., cDNA) and/or messenger RNA ofthe enzyme(s) so that the cell is capable of producing the enzyme(s).For example, nepenthesin can be produced by establishing host-vectorsystems in organisms such as Escherichia coli, Saccharomyces cerevisiae,Pichia pastoris, Lactobacillus, Bacilli, Aspergilli, and plant cellcultures, such as tobacco cells, etc.

Vectors and host cells, such as E. coli, comprising polynucleotides andcompositions containing any of the polynucleotides or polypeptides asdescribed herein are also provided.

In another aspect, provided is a method for producing recombinantNepenthes enzyme (nepenthesin I, nepenthesin II, and/or neprosin, or avariant thereof) comprising expressing in a chosen host organism anucleic acid sequence which encodes said enzyme, and inserting thenucleic acid sequence into an appropriately designed vector. In oneaspect, the recombinant enzyme is nepenthesin I or a variant thereof. Inone aspect, the recombinant enzyme is nepenthesin II or a variantthereof. In one aspect, the recombinant enzyme is neprosin or a variantthereof. In one aspect, the recombinant enzyme is a mixture ofnepenthesin I, nepenthesin II, and/or neprosin or variant thereof.

In another aspect, provided is a composition comprising recombinantnepenthesin such as nepenthesin I and/or nepenthesin II or a variantthereof. In one aspect, the recombinant nepenthesin is nepenthesin I ora variant thereof. In one aspect, the recombinant nepenthesin isnepenthesin II or a variant thereof. In one aspect, the recombinantnepenthesin is a mixture of nepenthesin I and nepenthesin II or variantsthereof.

In one aspect, this invention relates to a cDNA as described herein. Inone embodiment, this invention relates to a vector comprising a cDNA asdescribed herein. In a preferred embodiment, the vector is an expressionvector. In one embodiment, this invention relates to a cell expressingrecombinant nepenthesin I, recombinant nepenthesin II, recombinantneprosin, a variant or mixture thereof.

In some embodiments, biosynthesis of Nepenthes enzyme(s) can be achievedby transforming a cell with a vector comprising a cDNA that encodesnepenthesin I, for example the nucleotide sequence of SEQ ID NO. 4, SEQID NO. 5, SEQ ID NO.: 6, GenBank Accession No. JX494401, GenBankAccession No. AB114914, or GenBank Accession No. AB266803. In someembodiments, biosynthesis of nepenthesin can be achieved by transforminga cell with a vector comprising a sequence homologous to a cDNA whichencodes nepenthesin I, which sequence encodes a protein with proteaseactivity. The sequence may have at least about 60% homology to a cDNAthat encodes nepenthesin I. The sequence may have at least about 70%homology to a cDNA that encodes nepenthesin I. The sequence may have atleast about 80% homology to a cDNA that encodes nepenthesin I. Thesequence may have at least about 85% homology to a cDNA that encodesnepenthesin I. The sequence may have at least about 90% homology to acDNA that encodes nepenthesin I. The sequence may have at least about95% homology to a cDNA that encodes nepenthesin I. The sequence may haveat least about 96% homology to a cDNA that encodes nepenthesin I. Thesequence may have at least about 97% homology to a cDNA that encodesnepenthesin I. The sequence may have at least about 98% homology to acDNA that encodes nepenthesin I. The sequence may have at least about99% homology to a cDNA that encodes nepenthesin I. In a preferredembodiment, the sequence encodes a variant of nepenthesin I that retainsglutenase activity. In a particularly preferred embodiment, the sequenceencodes a variant of nepenthesin I that degrades at least one toxicgluten peptide.

In some embodiments, biosynthesis of Nepenthes enzyme(s) can be achievedby transforming a cell with a vector comprising a cDNA that encodesnepenthesin II, for example the nucleotide sequence of SEQ ID NO.: 8,SEQ ID NO.: 9, GenBank Accession No. JX494402 or GenBank Accession No.AB114915. In some embodiments, biosynthesis of nepenthesin can beachieved by transforming a cell with a vector comprising a sequencehomologous to a cDNA which encodes nepenthesin II, which sequenceencodes a protein with protease activity. The sequence may have at leastabout 60% homology to a cDNA that encodes nepenthesin II. The sequencemay have at least about 70% homology to a cDNA that encodes nepenthesinII. The sequence may have at least about 80% homology to a cDNA thatencodes nepenthesin II. The sequence may have at least about 85%homology to a cDNA that encodes nepenthesin II. The sequence may have atleast about 90% homology to a cDNA that encodes nepenthesin II. Thesequence may have at least about 95% homology to a cDNA that encodesnepenthesin II. The sequence may have at least about 96% homology to acDNA that encodes nepenthesin II. The sequence may have at least about97% homology to a cDNA that encodes nepenthesin II. The sequence mayhave at least about 98% homology to a cDNA that encodes nepenthesin II.The sequence may have at least about 99% homology to a cDNA that encodesnepenthesin II. In a preferred embodiment, the sequence encodes avariant of nepenthesin II that retains glutenase activity. In aparticularly preferred embodiment, the sequence encodes a variant ofnepenthesin H that degrades at least one toxic gluten peptide.

In some embodiments, biosynthesis of Nepenthes enzyme(s) can be achievedby transforming a cell with a vector comprising a cDNA that encodesneprosin, for example the nucleotide sequence of SEQ ID NO.: 2. In someembodiments, biosynthesis of neprosin can be achieved by transforming acell with a vector comprising a sequence homologous to a cDNA whichencodes neprosin, which sequence encodes a protein with proteaseactivity. The sequence may have at least about 60% homology to a cDNAthat encodes neprosin. The sequence may have at least about 70% homologyto a cDNA that encodes neprosin. The sequence may have at least about80% homology to a cDNA that encodes neprosin. The sequence may have atleast about 85% homology to a cDNA that encodes neprosin. The sequencemay have at least about 90% homology to a cDNA that encodes neprosin.The sequence may have at least about 95% homology to a cDNA that encodesneprosin. The sequence may have at least about 96% homology to a cDNAthat encodes neprosin. The sequence may have at least about 97% homologyto a cDNA that encodes neprosin. The sequence may have at least about98% homology to a cDNA that encodes neprosin. The sequence may have atleast about 99% homology to a cDNA that encodes neprosin. In a preferredembodiment, the sequence encodes a variant of neprosin that retainsprolyl endoprotease activity. In an especially preferred embodiment, thesequence encodes a variant of neprosin that retains glutenase activity.In a particularly preferred embodiment, the sequence encodes a variantof neprosin that degrades at least one toxic gluten peptide.

Without being bound by theory, it is believed that inflammatory responseto gluten in the intestines of affected individuals is due to theincomplete hydrolysis of gluten proteins, leading to the formation oftoxic (immunotoxic) gluten peptides. Several immunotoxic and/orpotentailly immunotoxic gluten peptides are known. These include, butare not limited to, the 33-mer (SEQ ID NO.: 15, LQLQPF(PQPQLPY)₃PQPQPF)and p31-49 (SEQ ID NO.: 16, LGQQQPFPPQQPYPQPQPF) from α-gliadin; Gly-156(SEQ ID NO.: 17, QQQQPPFSQQQQSPFSQQQQ) from low molecular weightglutenin; and the nonapeptide repeat (SEQ ID NO.: 18, GYYPTSPQQ) andhexapeptide repeat (SEQ ID NO.: 19, PGQGQQ) from high molecular weightglutenin.

In some embodiments, nepenthesin I, nepenthesin II, neprosin and/or avariant thereof is synthesized by transfecting, infecting, ortransforming a cell with one or more vectors comprising a cDNA sequenceof each desired enzyme. That is, a single cell, cell line, or organismmay be engineered so as to produce two or more enzymes. In someembodiments, the desired enzymes are synthesized by separate cells andcombined in the pharmaceutical composition. In a preferred embodiment,the recombinant nepenthesin I, nepenthesin II, neprosin and/or a variantthereof is not glycosylated. In one embodiment, the recombinantnepenthesin I, nepenthesin II, neprosin and/or a variant thereof has adifferent glycosylation pattern than the natural enzyme (i.e.,nepenthesin I, nepenthesin II, or neprosin isolated from a Nepenthesplant).

The synthetic (e.g., recombinant) Nepenthes enzyme(s) can beconcentrated or purified according to known methods, such as those forisolating Nepenthes enzyme(s) from the plant pitcher liquid.

In some embodiments, the protein product isolated from a natural sourceor a synthetic (e.g., recombinant) source comprises at least 20% byweight of at least one Nepenthes enzyme or a variant thereof. In someembodiments, the isolated protein product comprises at least about 50%,about 75%, about 90%, about 95% by weight of the Nepenthes enzyme orvariant thereof. In some embodiments, the isolated protein productcomprises at least 99% by weight of the Nepenthes enzyme or variantthereof.

In some embodiments, the recombinant Nepenthes enzyme or variant thereofcomprises substantially only recombinant nepenthesin or variant thereof.In some embodiments, the recombinant nepenthesin or variant thereofcomprises substantially only recombinant nepenthesin I or variantthereof. In some embodiments, the recombinant nepenthesin or variantthereof comprises substantially only nepenthesin II or variant thereof.In some embodiments, the recombinant nepenthesin or variant thereofcomprises nepenthesin I and nepenthesin II, or variant thereof. In someembodiments, the recombinant nepenthesin or variant thereof comprises aratio of nepenthesin I to nepenthesin II (or variant of each thereof) ofat least about 100:1. In some embodiments, the recombinant nepenthesincomprises a ratio of nepenthesin I to nepenthesin II of at least about90:1. In some embodiments, the recombinant nepenthesin comprises a ratioof nepenthesin I to nepenthesin II of at least about 70:1. In someembodiments, the recombinant nepenthesin comprises a ratio ofnepenthesin I to nepenthesin II of at least about 60:1. In someembodiments, the recombinant nepenthesin comprises a ratio ofnepenthesin I to nepenthesin II of at least about 50:1. In someembodiments, the recombinant nepenthesin comprises a ratio ofnepenthesin I to nepenthesin II of at least about 40:1. In someembodiments, the recombinant nepenthesin comprises a ratio ofnepenthesin I to nepenthesin II of at least about 30:1. In someembodiments, the recombinant nepenthesin comprises a ratio ofnepenthesin I to nepenthesin II of at least about 20:1. In someembodiments, the recombinant nepenthesin comprises a ratio ofnepenthesin I to nepenthesin II of at least about 10:1. In someembodiments, the recombinant nepenthesin comprises a ratio ofnepenthesin I to nepenthesin II of at least about 5:1. In someembodiments, the recombinant nepenthesin comprises a ratio ofnepenthesin I to nepenthesin II of at least about 4:1. In someembodiments, the recombinant nepenthesin comprises a ratio ofnepenthesin I to nepenthesin II of at least about 3:1. In someembodiments, the recombinant nepenthesin comprises a ratio ofnepenthesin I to nepenthesin II of at least about 2:1. In someembodiments, the recombinant nepenthesin comprises a ratio ofnepenthesin I to nepenthesin II of at least about 1:1. In someembodiments, the recombinant nepenthesin comprises a ratio ofnepenthesin I to nepenthesin II of at least about 1:2. In someembodiments, the recombinant nepenthesin comprises a ratio ofnepenthesin I to nepenthesin II of at least about 1:3. In someembodiments, the recombinant nepenthesin comprises a ratio ofnepenthesin I to nepenthesin II of at least about 1:4. In someembodiments, the recombinant nepenthesin comprises a ratio ofnepenthesin I to nepenthesin II of at least about 1:5. In someembodiments, the recombinant nepenthesin comprises a ratio ofnepenthesin I to nepenthesin II of at least about 1:10. In someembodiments, the recombinant nepenthesin comprises a ratio ofnepenthesin I to nepenthesin II of at least about 1:20. In someembodiments, the recombinant nepenthesin comprises a ratio ofnepenthesin I to nepenthesin II of at least about 1:30. In someembodiments, the recombinant nepenthesin comprises a ratio ofnepenthesin I to nepenthesin II of at least about 1:40. In someembodiments, the recombinant nepenthesin comprises a ratio ofnepenthesin I to nepenthesin II of at least about 1:50. In someembodiments, the recombinant nepenthesin comprises a ratio ofnepenthesin I to nepenthesin II of at least about 1:60. In someembodiments, the recombinant nepenthesin comprises a ratio ofnepenthesin I to nepenthesin II of at least about 1:70. In someembodiments, the recombinant nepenthesin comprises a ratio ofnepenthesin I to nepenthesin II of at least about 1:80. In someembodiments, recombinant nepenthesin comprises a ratio of nepenthesin Ito nepenthesin II of at least about 1:90. In some embodiments, therecombinant nepenthesin comprises a ratio of nepenthesin I tonepenthesin II of at least about 1:100.

In some embodiments, the recombinant Nepenthes enzyme or variant thereofcomprises substantially only recombinant neprosin or variant thereof. Insome embodiments, the recombinant Nepenthes enzyme or variant thereofcomprises neprosin and nepenthesin or variant thereof. In someembodiments, the recombinant Nepenthes enzyme or variant thereofcomprises neprosin and nepenthesin I or variant thereof. In someembodiments, the recombinant Nepenthes enzyme or variant thereofcomprises neprosin and nepenthesin II or variant thereof. In someembodiments, the recombinant Nepenthes enzyme or variant thereofcomprises neprosin, nepenthesin I and nepenthesin II, or variantthereof. In some embodiments, the recombinant Nepenthes enzyme orvariant thereof comprises a ratio of neprosin to nepenthesin (or variantof each thereof) of at least about 100:1. In some embodiments, therecombinant Nepenthes enzyme comprises a ratio of neprosin tonepenthesin of at least about 90:1. In some embodiments, the recombinantNepenthes enzyme comprises a ratio of neprosin to nepenthesin of atleast about 70:1. In some embodiments, the recombinant Nepenthes enzymecomprises a ratio of neprosin to nepenthesin of at least about 60:1. Insome embodiments, the recombinant Nepenthes enzyme comprises a ratio ofneprosin to nepenthesin of at least about 50:1. In some embodiments, therecombinant Nepenthes enzyme comprises a ratio of neprosin tonepenthesin of at least about 40:1. In some embodiments, the recombinantNepenthes enzyme comprises a ratio of neprosin to nepenthesin of atleast about 30:1. In some embodiments, the recombinant Nepenthes enzymecomprises a ratio of neprosin to nepenthesin of at least about 20:1. Insome embodiments, the recombinant Nepenthes enzyme comprises a ratio ofneprosin to nepenthesin of at least about 10:1. In some embodiments, therecombinant Nepenthes enzyme comprises a ratio of neprosin tonepenthesin of at least about 5:1. In some embodiments, the recombinantNepenthes enzyme comprises a ratio of neprosin to nepenthesin of atleast about 4:1. In some embodiments, the recombinant Nepenthes enzymecomprises a ratio of neprosin to nepenthesin of at least about 3:1. Insome embodiments, the recombinant Nepenthes enzyme comprises a ratio ofneprosin to nepenthesin of at least about 2:1. In some embodiments, therecombinant Nepenthes enzyme comprises a ratio of neprosin tonepenthesin of at least about 1:1. In some embodiments, the recombinantNepenthes enzyme comprises a ratio of neprosin to nepenthesin of atleast about 1:2. In some embodiments, the recombinant Nepenthes enzymecomprises a ratio of neprosin to nepenthesin of at least about 1:3. Insome embodiments, the recombinant Nepenthes enzyme comprises a ratio ofneprosin to nepenthesin of at least about 1:4. In some embodiments, therecombinant Nepenthes enzyme comprises a ratio of neprosin tonepenthesin of at least about 1:5. In some embodiments, the recombinantNepenthes enzyme comprises a ratio of neprosin to nepenthesin of atleast about 1:10. In some embodiments, the recombinant Nepenthes enzymecomprises a ratio of neprosin to nepenthesin of at least about 1:20. Insome embodiments, the recombinant Nepenthes enzyme comprises a ratio ofneprosin to nepenthesin of at least about 1:30. In some embodiments, therecombinant Nepenthes enzyme comprises a ratio of neprosin tonepenthesin of at least about 1:40. In some embodiments, the recombinantNepenthes enzyme comprises a ratio of neprosin to nepenthesin of atleast about 1:50. In some embodiments, the recombinant Nepenthes enzymecomprises a ratio of neprosin to nepenthesin of at least about 1:60. Insome embodiments, the recombinant Nepenthes enzyme comprises a ratio ofneprosin to nepenthesin of at least about 1:70. In some embodiments, therecombinant Nepenthes enzyme comprises a ratio of neprosin tonepenthesin of at least about 1:80. In some embodiments, recombinantNepenthes enzyme comprises a ratio of neprosin to nepenthesin of atleast about 1:90. In some embodiments, the recombinant Nepenthes enzymecomprises a ratio of neprosin to nepenthesin of at least about 1:100.

In some embodiments, the protein product isolated from a natural sourceor a synthetic source comprises an amino acid that is at least about 70%homologous to the amino acid sequence of Nepenthes nepenthesin I (e.g.,SEQ ID NO.: 5; SEQ ID NO.: 6; SEQ ID NO.: 7; SEQ ID NO.: 21). In oneembodiment, the protein product retains protease activity. The proteinmay be at least about 80% homologous to Nepenthes nepenthesin I. Theprotein may be at least about 85% homologous to Nepenthes nepenthesin I.The protein may be at least about 90% homologous to Nepenthesnepenthesin I. The protein may be at least about 95% homologous toNepenthes nepenthesin I. The protein may be at least about 96%homologous to Nepenthes nepenthesin I. The protein may be at least about97% homologous to Nepenthes nepenthesin I. The protein may be at leastabout 98% homologous to Nepenthes nepenthesin I. The protein may be atleast about 99% homologous to Nepenthes nepenthesin I.

In some embodiments, the protein product isolated from a natural sourceor a synthetic source comprises a protein that is at least about 70%homologous to Nepenthes nepenthesin II (e.g., SEQ ID NO.: 8; SEQ ID NO.:9; SEQ ID NO.: 20). In one embodiment, the protein product retainsprotease activity. The protein may be at least about 80% homologous toNepenthes nepenthesin II. The protein may be at least about 85%homologous to Nepenthes nepenthesin II. The protein may be at leastabout 90% homologous to Nepenthes nepenthesin II. The protein may be atleast about 95% homologous to Nepenthes nepenthesin II. The protein maybe at least about 96% homologous to Nepenthes nepenthesin II. Theprotein may be at least about 97% homologous to Nepenthes nepenthesinII. The protein may be at least about 98% homologous to Nepenthesnepenthesin II. The protein may be at least about 99% homologous toNepenthes nepenthesin II.

In some embodiments, the protein product isolated from a natural sourceor a synthetic source comprises a protein that is at least about 70%homologous to Nepenthes neprosin (e.g., SEQ ID NO.: 1). In oneembodiment, the protein product retains protease activity. The proteinmay be at least about 80% homologous to Nepenthes neprosin. The proteinmay be at least about 85% homologous to Nepenthes neprosin. The proteinmay be at least about 90% homologous to Nepenthes neprosin. The proteinmay be at least about 95% homologous to Nepenthes neprosin. The proteinmay be at least about 96% homologous to Nepenthes neprosin. The proteinmay be at least about 97% homologous to Nepenthes neprosin. The proteinmay be at least about 98% homologous to Nepenthes neprosin. The proteinmay be at least about 99% homologous to Nepenthes neprosin.

In some embodiments, the protein product isolated from a natural sourceor a synthetic source comprises nepenthesin or a variant thereof with atleast about 10% of the original protease activity of Nepenthesnepenthesin I. In some embodiments, the protein product comprisesnepenthesin or a variant thereof with at least about 20% of the originalprotease activity of nepenthesin I. In some embodiments, the proteinproduct comprises nepenthesin or a variant thereof with at least about30% of the original protease activity of nepenthesin I. In someembodiments, the protein product comprises nepenthesin or a variantthereof with at least about 40% of the original protease activity ofnepenthesin I. In some embodiments, the protein product comprisesnepenthesin or a variant thereof with at least about 50% of the originalprotease activity of nepenthesin I. In some embodiments, the proteinproduct comprises nepenthesin or a variant thereof with at least about60% of the original protease activity of nepenthesin I. In someembodiments, the protein product comprises nepenthesin or a variantthereof with at least about 70% of the original protease activity ofnepenthesin I. In some embodiments, the protein product comprisesnepenthesin or a variant thereof with at least about 80% of the originalprotease activity of nepenthesin I. In some embodiments, the proteinproduct comprises nepenthesin or a variant thereof with at least about90% of the original protease activity of nepenthesin I. In someembodiments, the protein product comprises nepenthesin or a variantthereof with greater than about 100% of the original protease activityof nepenthesin I.

In some embodiments, the protein product isolated from a natural sourceor a synthetic source comprises nepenthes in or a variant thereof withat least about 10% of the original protease activity of Nepenthesnepenthesin II. In some embodiments, the protein product comprisesnepenthesin or a variant thereof with at least about 20% of the originalprotease activity of nepenthesin II. In some embodiments, the proteinproduct comprises nepenthesin or a variant thereof with at least about30% of the original protease activity of nepenthesin II. In someembodiments, the protein product comprises nepenthesin or a variantthereof with at least about 40% of the original protease activity ofnepenthesin II. In some embodiments, the protein product comprisesnepenthesin or a variant thereof with at least about 50% of the originalprotease activity of nepenthesin II. In some embodiments, the proteinproduct comprises nepenthesin or a variant thereof with at least about60% of the original protease activity of nepenthesin II. In someembodiments, the protein product comprises nepenthesin or a variantthereof with at least about 70% of the original protease activity ofnepenthesin II. In some embodiments, the protein product comprisesnepenthesin or a variant thereof with at least about 80% of the originalprotease activity of nepenthesin II. In some embodiments, the proteinproduct comprises nepenthesin or a variant thereof with at least about90% of the original protease activity of nepenthesin II. In someembodiments, the protein product comprises nepenthesin or a variantthereof with greater than about 100% of the original protease activityof nepenthesin II.

In some embodiments, the protein product isolated from a natural sourceor a synthetic source comprises neprosin or a variant thereof with atleast about 10% of the original protease activity of Nepenthes neprosin.In some embodiments, the protein product comprises neprosin or a variantthereof with at least about 20% of the original protease activity ofneprosin. In some embodiments, the protein product comprises neprosin ora variant thereof with at least about 30% of the original proteaseactivity of neprosin. In some embodiments, the protein product comprisesneprosin or a variant thereof with at least about 40% of the originalprotease activity of neprosin. In some embodiments, the protein productcomprises neprosin or a variant thereof with at least about 50% of theoriginal protease activity of neprosin. In some embodiments, the proteinproduct comprises neprosin or a variant thereof with at least about 60%of the original protease activity of neprosin. In some embodiments, theprotein product comprises neprosin or a variant thereof with at leastabout 70% of the original protease activity of neprosin. In someembodiments, the protein product comprises neprosin or a variant thereofwith at least about 80% of the original protease activity of neprosin.In some embodiments, the protein product comprises neprosin or a variantthereof with at least about 90% of the original protease activity ofneprosin. In some embodiments, the protein product comprises neprosin ora variant thereof with greater than about 100% of the original proteaseactivity of neprosin.

Unless stated otherwise, the abbreviations used throughout thespecification have the following meanings:

-   -   g=gram    -   kDa=kiloDalton    -   kg=kilogram    -   L=liter    -   LC=liquid chromatography    -   mg=milligram    -   min=minute    -   mL=milliliter    -   mM=millimolar    -   MS=mass spectrometry    -   nM=nanomolar    -   pM=picomolar    -   s.d.=standard deviation    -   μCi=microcurie    -   μg=microgram    -   μL=microliter    -   μM=micromolar    -   μm=micrometer    -   ° C.=degree Celsius

These one-letter symbols have the following meaning when representingamino acids:

-   -   A=Alanine    -   R=Arginine    -   N=Asparagine    -   D=Aspartic acid    -   C=Cysteine    -   E=Glutamic acid    -   Q=Glutamine    -   G=Glycine    -   H=Histidine    -   I=Isoleucine    -   L=Leucine    -   K=Lysine    -   M=Methionine    -   F=Phenylalanine    -   P=Proline    -   S=Serine    -   T=Threonine    -   W=Tryptophan    -   Y=Tyrosine    -   V=Valine

EXAMPLES Example 1 Nepenthesin Extract Preparation Chemicals

Water and acetonitrile, HPLC grade form Burdick and Jackson, werepurchased from VWR. Formic acid, Tris, and glycine were purchased fromSigma Aldrich:

Plant Culture

Transplants of Nepenthes rafflesiana, Nepenthes ampularia, Nepenthesmirabilis, and Nepenthes globosa were purchased from Keehns Carnivores(www.keehnscarnivores.ca). These were potted with wood bark, perlite,peat moss and humus (40, 35, 10, 5% respectively). Growth conditionsinvolved 14 hours of light per day, 80% humidity and temperature in the23° C. to 28° C. range with 2 to 3 waterings a week. Upon pitchermaturity, plants were fed with one or two Drosophila per pitcher and thepitcher fluid harvested one week later. Pitchers and their secretionswere left to recover for one week prior to a second round of feeding andextraction.

Extract Preparation

Pitcher fluid was collected from all four species of plants andcombined. The crude pitcher fluid was first clarified through a 0.22 μmfilter, then concentrated 80 to 100 fold using an Amicon Ultracentrifugal 10 kDa molecular weight cut-off filter (both fromMillipore). Prior to use in digestions, the concentrate wasacid-activated with 100 mM Glycine HCl (pH 2.5) for 3 hours, then washed3× with 100 mM Glycine-HCl (pH 2.5) in the filtration device, using 10×fluid volume for each wash. The final isolate was then rediluted to an11× concentration based on the original sampling of pitcher fluid.

Characterization of Pitcher Fluid Extract

The fluidic secretions of the pitcher plant were concentrated and thedigestion enzymes activated by pH reduction (pH 2.5). The impact of theenrichment process and the activation on the fluid proteome wasdetermined using proteomics methods. First, to confirm the presence ofnepenthesin enzyme, the inactive concentrate was separated by SDS-PAGE.Seven contiguous gel zones with very faint coomassie staining weredigested with trypsin and analyzed by nanoLC-MS/MS using standardmethods. This is not expected to be a complete catalog of the activatedfluid proteome, but the analysis confirmed the presence of the asparticprotease nepenthesin I/II, as well as a glucanase, chitinase,carboxypeptidase and peroxidase of plant origin, plus modest levels ofdrosophila and bacterial contamination. The low complexity of the fluidproteome is consistent with recent analyses, Hatano N, Hamada T (2012)Proteomic analysis of secreted protein induced by a component of prey inpitcher fluid of the carnivorous plant Nepenthes alata. Journal ofProteomics 3; 75(15):4844-52 (Epub Jun. 15, 2012), but nepenthesin-I wasfound distributed over a much wider mass range in this analysis (40-70kDa).

The acid-activated fluid was then processed and analyzed in a similarfashion. The activation process reduced the overall protein yield, andalso appeared to simplify the composition. Aside from nepenthesin-I,only minor contamination from keratin and actin were in evidence. Theseanalyses point to the low complexity of the enriched fluid, wherenepenthesin is the major component. The total protein concentration ofthe activated and 80× enriched fluid was measured by a BCA assay to be22 ng/μL. This value is consistent with an earlier study describingenrichment of the fluid. Tokes Z A, et al., Digestive-Enzymes Secretedby Carnivorous Plant Nepenthes-Macferlanei-L. Planta 119(1):39-46(1974).

Example 2 Nepenthesin Extract Purification Purification of Extract

Sepharose-immobilized pepstatin in a 50×2 cm ID column was equilibratedin 20 mM Glycine-HCl, pH 2.5-3. The filtered pitcher fluid (prepared asdescribed in Example 1) was cycled twice through the column, and thecolumn washed with 100 mL equilibration buffer (20 mM glycine HCl, pH2.5). The column was eluted with 100 mM ammonium bicarbonate pH 8.7 andfractions collected. In order to preserve maximum the enzyme activity,the pH was decreased to 4 right after fraction collection with 2 Mglycine HCl, pH 2.5. Activity was verified using a digestion assay, andthe most active fractions combined and concentrated to approximately80×, based on original fluid volume.

The only endoproteases found at detectable levels in the Nepenthes fluidand/or extract are aspartic proteases and prolyl endoprotease.

Example 3 Recombinant Nepenthesin I

The gene for nepenthesin I (SEQ ID NO: 4; encoding amino acid residues20-413, from N. gracilis, without the plant signal sequence) wasprepared from nepenthesin I cDNA, and placed between NdeI and HindIIIrestriction sites. This sequence was cloned into pET21a, using T4 DNAligase (1 U) (New England Bio, NEB), T4 DNA ligase buffer (NEB), ATP(0.5 mM) (NEB), 0.5 μg pET21a vector and 2 μg of the nepenthesin I cDNA.This was incubated at 18° C. for 4 hours. The ligation mixture (5 μL)was added to 200 μL of NovaBlue competent cells and incubated on ice for15 minutes. Cells were transformed by heat shock (45 seconds at 42° C.,then immediately on ice, with 1 ml of LB medium) and incubated for 1hour at 37° C., and plated with antibiotics (tetracycline andampicillin). After confirming gene presence in several white colonies, arepresentative colony was chosen for maxiprep. The resulting recombinantplasmid pET21a/R.NepI was transformed into E. coli C41 by heat-shock asabove, for expression under induction by IPTG. Here, cells were grown upto an OD₆₆₀ of 0.6 and induced with 0.1 mM IPTG for four hours at 37° C.The expressed protein went to inclusion bodies.

Inclusion bodies were isolated as follows. Cells were centrifuged,sucrose lysis buffer was added (25% saccharose, 50 mM TrisCl pH 7.4, 1mM EDTA, 1 mM NaN₃, and protease inhibitors), and the cells weresubjected to four rounds of freeze/thaw and sonication. This wasfollowed by the addition of DNAse and RNAse for a 30 min. incubation atroom temperature. The preparation was centrifuged (˜15 min. at 5000×g)to pellet the inclusion bodies and membrane fragments. This pellet wasresuspended in Triton buffer (50 mM TrisCl pH 7.4, 10 mM NaCl, 1 mMβ-mercaptoethanol, 1 mM NaN₃, 0.5% Triton X100+protease inhibitors) andsonication performed on ice. This was once again centrifuged, to pelletthe inclusion bodies, and the pellet was washed twice on ice (withmixing and sonication) in a buffer free of Triton (50 mM TrisCl pH 7.4,10 mM NaCl, 1 mM β-mercaptoethanol, 1 mM NaN₃, protease inhibitors).

The protein pellet was then subjected to refolding. One g of inclusionbodies was suspended into 1 L of 50 mM CAPS pH 10.5, 8 M urea, 1 mMEDTA, 1 mM glycine, 500 mM NaCl, 300 mM β-mercaptoethanol and shaken for1 hr. The suspension was dialysed against 50 mM Tris, pH 11, twice for 1hour at a time, followed by one day of dialysis against 50 mM Tris, pH7.5, and finally, dialysis against phosphate buffer with 300 mM NaCl, pH7.0.

The solution was centrifuged at high speed (10000×g for 15 min.) toremove any un-refolded protein, and the supernatant filtered through a0.22 μm membrane. Nepenthesin I was activated at pH 2.5 (glycine-HCl)overnight at 4° C. Yields range from 10 to 100 mg of folded, activatedprotein, starting from 1 L of cell culture.

Example 4 Recombinant Nepenthesin II

The cDNA of nepenthesin II (see SEQ ID NO.: 14) from N. gracilis,without the plant signal sequence) was used to prepare nepenthesin IIcDNA. This sequence was cloned into pET21a between NdeI and HindIIIrestriction sites, using T4 DNA ligase (1 U) (New England Bio, NEB), T4DNA ligase buffer (NEB), ATP (0.5 mM) (NEB), 0.5 μg pET21a vector and 2μg of the nepenthesin H cDNA. This was incubated at 18° C. for 4 hours.The ligation mixture (5 μL) was added to 200 μL of NovaBlue competentcells and incubated on ice for 15 minutes. Cells were transformed byheat shock (45 seconds at 42° C., then immediately on ice, with 1 ml ofLB medium) and incubated for 1 hour at 37° C., and plated withantibiotics (tetracycline and ampicillin). After confirming genepresence in several white colonies, a representative colony was chosenfor maxiprep. The resulting recombinant plasmid pET21a/R.NepI wastransformed into E. coli C41 by heat-shock as above, for expressionunder induction by IPTG. Here, cells were grown up to an OD₆₆₀ of 0.6and induced with 0.1 mM IPTG for four hours at 37° C. The expressedprotein went to inclusion bodies.

Inclusion bodies were isolated as follows. Cells were centrifuged,sucrose lysis buffer was added (25% saccharose, 50 mM TrisCl pH 7.4, 1mM EDTA, 1 mM NaN₃, and protease inhibitors), and the cells weresubjected to four rounds of freeze/thaw and sonication. This wasfollowed by the addition of DNAse and RNAse for a 30 min. incubation atroom temperature. The preparation was centrifuged (˜15 min. at 5000×g)to pellet the inclusion bodies and membrane fragments. This pellet wasresuspended in Triton buffer (50 mM TrisCl pH 7.4, 10 mM NaCl, 1 mMβ-mercaptoethanol, 1 mM NaN₃, 0.5% Triton X100+protease inhibitors) andsonication performed on ice. This was once again centrifuged, to pelletthe inclusion bodies, and the pellet was washed twice on ice (withmixing and sonication) in a buffer free of Triton (50 mM TrisCl pH 7.4,10 mM NaCl, 1 mM β-mercaptoethanol, 1 mM NaN₃, protease inhibitors).

The protein pellet was then subjected to refolding. One g of inclusionbodies was suspended into 1 L of 50 mM CAPS pH 10.5, 8 M urea, 1 mMEDTA, 1 mM glycine, 500 mM NaCl, 300 mM β-mercaptoethanol and shaken for1 hr. The suspension was dialysed against 50 mM Tris pH 11 twice for 1hour at a time, followed by one day of dialysis against 50 mM Tris pH7.5, and finally, dialysis against phosphate buffer with 300 mM NaCl, pH7.0.

The solution was centrifuged at high speed (10000×g for 15 min.) toremove any un-refolded protein, and the supernatant filtered through a0.22 μm membrane. Nepenthesin II was activated at pH 2.5 (glycine-HCl)overnight at 4° C. Yields range from 10 to 100 mg of folded, activatedprotein, starting from 1 L of cell culture.

Example 5 Glycosylation of Nepenthes Enzymes

Recombinant production of nepenthesin I (A) and II (C) from refolding ofpurified E. coli inclusion bodies is shown in FIG. 2. Each step of therefolding procedure was monitored and is shown as: total solubilizedprotein from purified E. coli inclusion bodies (Lane 1), refoldednepenthesin after final dialysis (lane 2), 24-hour acid activation (100mM glycine-HCl, pH 2.5) of refolded product (lane 3). MALDI-TOF MSanalysis was performed on the 24-hour acid activated nepenthesin I (B)and II (D) enzymes. LC-MS/MS analyses of in-gel digests of theacid-activated bands (A and C, lanes 3) confirmed the presence of purenepenthesin I and II respectively.

MALDI-TOF analyses of natural nepenthesins (pooled from 2-3 species) wasperformed. Results are shown in FIG. 3. The mass at 37,200 is believedto be nepenthesin II and the mass at 38,951 to be nepenthesin I. Eitherway, they are different than the masses of the recombinant enzymes, asshown in Table 1.

TABLE 1 Mass of Recombinant v. Natural Nepenthesins Nepenthesin 1 Mass(Daltons)* Nepenthesin II Mass (Daltons)* recombinant 37,460 recombinant37,506 natural 38,949 natural 37,199 Difference: 1,489 −307 *1 Dalton issubtracted for the proton added by MALDI.

Without being bound by theory, we believe that this confirms nepenthesinI is glycosylated in nature. The active, mature enzyme of recombinantnepenthesin II is larger than what exists in nature. It remains possiblethat natural nepenthesin II is even smaller in protein sequence but hassome minor glycosyation. The masses of the natural enzymes reportedherein differ from Athauda et al. likely because mass spec is a moreaccurate technique than SDS PAGE for determining the mass of a molecule.

Example 6 Comparison of Nepenthes Enzymes with Pepsin

SDS-PAGE was performed on gliadin digested by the indicated enzyme.SDS-PAGE roughly profiles proteins according to molecular weight.Gliadin digestion with pepsin, purified Nepenthes extract, orrecombinant nepenthesin II was performed at a substrate to enzyme ratioof approximately 100:1. Gliadin (5 mg) was incubated with the indicatedpreparation at 37° C. for 2 hr. FIG. 4 shows an SDS-PAGE gel of gliadindigestion by recombinant nepenthesin II, Nepenthes extract, or pepsin.The gel shows that digestion of gliadin by recombinant nepenthesin IIresults in a different digestion pattern and digestion into smallerpeptides than does pepsin. This is particularly noticeable in the boxedareas of the gel. Nepenthes extract is so efficient at degrading gliadinthat no residual gliadin protein is observed in this region.

Table 2 indicates preferred, low probability, and forbidden residues forC-terminal cleavage by pepsin, recombinant nepenthesin I and II, andNepenthes extract. C-terminal cleavage specificity, the classic wayenzymes are classified, is summarized based on a large collection ofprotein substrates. The nepenthesins are quite different from pepsin incleavage specificity, indicating that nepenthesin and pepsin are verydifferent enzymes. The pepsin data provided in Table 2 is summarizedfrom the literature (e.g. “Determining the Specificity of Pepsin forProteolytic Digestion”, a thesis by Melissa Palashoff available at:books.google.ca/books?id=7OlnU4-6T-wC&printsec=frontcover#v=onepage&q&f=false).Nepenthes enzyme data is summarized from digestions studies such as thatdescribed in U.S. Patent Application Publication No. 2014/0186330, whichis incorporated herein by reference in its entirety.

TABLE 2 C-terminal Cleavage Nepenthesin I and Pepsin II Nepenthesextract Preferred F, L, M F, L, M, K, R, D, F, L, M, K, R, E, C, Y, A D,E, C, Y Low W, C, Y, D, E, G, W, G, N, Q, V, T H, I, A, P, N, Qprobability Q, N, S, T “Forbidden” I, V, K, R, P, H, G G, I, S, P G, S,T, W, V

LC-MS assay was performed to determine the ability of each enzyme tocleave the 33-mer toxic gluten peptide. 33-mer was incubated with theindicated enzyme for 0.5 h at a 100:1 ratio (substrate:enzyme), and theamount of undigested 33-mer determined relative to a standard, followingcommon practice. Data is provided as percent of the control (33-mer withno enzyme added).

Table 3 provides the digestion of the pepsin-resistant fragment fromgluten protein that is called the “33-mer”(LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF) SEQ ID. 15. This sequence isstrongly linked to celiac disease and is often termed a toxic glutenpeptide. Like the whole gluten proteins themselves, the 33-mer is richin Q, P and L amino acids. Extended digestion times using just pepsindid not have much of an effect on this peptide—it was very resistant topepsin digestion. In contrast, nepenthesin I, nepenthesin II and thehigh molecular weight fraction (>approx. 10 kDa) of Nepenthes extract(fluid) exhibited the ability to digest this peptide. Data are providedas % of control (33-mer with no enzyme).

TABLE 3 33-mer Digestion Relative Peak Area Enzyme for Digestion (%)Control 100.0 Nepenthes fluid 0.0 Nepenthesin I 78.7 Nepenthesin II 34.0Pepsin 93.2

Gliadin protein slurry (5 mg gluten) was incubated with 40 or 200 μg ofrecombinant nepenthesin I or recombinant nepenthesin II, or 40 μg ofpepsin and examined for degree of digestion (as determined by the degreeof cloudiness of the relative solutions). Increasing amounts of pepsinhave no effect on the cloudiness of the slurry (data not shown). FIG. 5Ais an image of vials containing gliadin slurry and the indicated amountof recombinant nepenthesin I, recombinant nepenthesin II, or pepsin.FIG. 5B is similar, but used 5, 20, or 100 μg of Nepenthes extract. Thevials incubated with nepenthesin or Nepenthes extract were less cloudythan the pepsin vial, showing more vigorous digestion of gliadin.

These data show that the gliadin protein digests are different betweenNepenthes enzymes and pepsin at the gel level (which shows the “larger”digestion products), the peptide level (processing of the 33-mer), andat the slurry level (clarifying the solution). Pepsin, neprosin, andnepenthesin are very different proteins with distinct cleavagespecificities, particularly with regard to gluten proteins. Simply put,pepsin does not adequately digest gluten in a manner to avoid glutentoxicity whereas the Nepenthes enzymes do.

Example 7 Digestion of Gliadin by Nepenthes Extract

Digestions of gliadin by nepenthesin were performed in solution using aLEAP HTX-PAL autosampler and dispensing system designed forhydrogen/deuterium exchange (HDX) applications. Data were collectedusing an AB Sciex Triple-TOF 5600 QqTOF mass spectrometer. Peptides wereidentified using Mascot (v2.3) from MS/MS data. Briefly, 12 pmol ofcrude gliadin (purchased from Sigma Aldrich) were mixed with 2 μL of100× concentrated extract, produced as described in Example 1. Afterdigestion the entire volume was injected into a reversed-phase LC systemconnected to the mass spectrometer. The peptides were trapped on a 7 cm,150 μm i.d. Magic C18 column and eluted with an acetonitrile gradientfrom 10% to 40% in 10 or 30 minutes. Peptides detected in these analyseswere selected for CID fragmentation in multiple information-dependentacquisitions of MS/MS spectra. Spectra were searched against a miniaturedatabase containing the sequences for all identified wheat gliadin (α,β, γ, ω) proteins plus the low and high molecular weight glutenin.

FIG. 6 shows the average length of all peptides identified from thenepenthes extract digestion of gliadin from wheat, using LC-MS/MS, after1, 5, 10, 15, 30, 60, 130, 360 or 810 minutes at 37° C. A 95% confidencecut-off (p<0.05) on the scores were used to reduce false positiveidentifications. Relative standard deviation of the peptide length isshown in the inset figure.

FIG. 7 displays the number of peptides identified by LC-MS/MS after 1,5, 10, 15, 30, 60, 130, 360 or 810 minutes digestion at 37° C., groupedby length. Data as in FIG. 6.

FIG. 8 displays the same data as in FIG. 6, as a probability ofobtaining a certain length after 10, 60, 120, 360 or 810 minutesdigestion at 37° C.

For digest mapping, gliadin digestion was performed as described above,except that the substrate to enzyme ratio was approximately 1000:1.Gliadin was digested at 37° C. for 2 hr with nepenthesin extract,purified nepenthesin extract, or recombinant nepenthesin I.

The P1 cleavage preference of recombinant nepenthesin I is very similarto that of the concentrated fluid extract, as well as the purifiedfraction of the extract (FIG. 9A). Surprisingly, the extract showed ahigher preference for glutamine than either the purified extract orrecombinant nepenthesin I.

The P1′ cleavage preference of recombinant nepenthesin I is very similarto that of the concentrated fluid extract, as well as the purifiedfraction of the extract (FIG. 9B). Surprisingly, the extract showed ahigher preference for proline than either the purified extract orrecombinant nepenthesin I.

The extract contains nepenthesin I, nepenthesin II, and neprosin, butthe purification strategy recovers more nepenthesin I than the other twoenzymes. Without wishing to be bound by theory, it is believed that theheightened cleavage at the PI glutamine position and the PI′ prolineposition by the extract are due to neprosin, nepenthesin II, and/orsynergy between two or more of the enzymes.

Example 8 Preparation of Neprosin Extract

Neprosin was extracted from Nepenthes sp. digesting fluid. The fluid wascollected from the plant pitcher 5 days after feeding with frozen fruitflies. The collected liquid was filtered to removed dead insects andrepeatedly washed with 20 mM ammonium acetate pH 5.0 by multipleconcentration/filtration cycles through a 10 kDa molecular weightcut-off membrane.

Neprosin was partially purified away from nepenthesin on a mono P 5/50GL column. 5 mL of 1.5× concentrated fluid was injected onto the mono Pcolumn equilibrated at low ionic strength (20 mM Ammonium acetate pH 6).The proteins were eluted with a 40 min NaCl gradient (0 to 1M) at 0.5ml/min. The fractions were collected every 0.5 ml. Neprosin activity wastested in each fraction by digesting an intrinsically-disorderedproline-rich protein, APLF. The peptides generated were separated on aC8 column and analyzed by LC-MS/MS on a tripleToF 5600 (AB Sciex).Fractions 19-22 were enriched for neprosin (FIG. 10) and are termed thecrude neprosin extract; neprosin is distinct from nepenthesin, which wasenriched in later fractions.

Example 9 Efficacy of Nepenthes Enzymes in Inhibiting Inflammation inthe Intestines of Gluten-Intolerant Mice

Objective:

To test the efficacy of in vitro digestion of gliadin using Nepenthesextract or recombinant nepenthesin II in preventing in vivogliadin-induced damage using gliadin-sensitized NOD-DQ8 mice.

Experimental Design:

NOD DQ8 mice were sensitized with cholera toxin (CT) and gliadin tobreak oral tolerance to gliadin. Negative controls were treated with CTand gliadin, but left free of subsequent oral gliadin challenges.Gliadin challenges were performed with a porcine protease (pepsin)digest of gliadin containing a variety of toxic and immunogenic derivedpeptides. Treatment groups were challenged with gliadin predigested withNepenthes extract or recombinant nepenthesin II (for 90 minutes at 37degrees Celsius). It is hypothesized that Nepenthes extract- orrecombinant nepenthesin II-gliadin digests will be less immunogenic invivo than pepsin-gliadin digests.

Groups:

Positive Control (n=8):

Sensitized and gliadin challenged. Mice were sensitized with choleratoxin (CT) and pepsin gliadin (P-G) (1× per week for 3 weeks). Duringthe experimental period, mice were gavaged with P-gliadin (3× per weekfor 3 weeks).

Negative Control (n=8):

Sensitized (then gliadin free). Mice were sensitized with cholera toxin(CT) and pepsin gliadin (P-G) (1× per week for 3 weeks). During theexperimental period, mice were gavaged with vehicle (3× per week for 3weeks).

Treatment 1 (n=8):

Nepenthes extract. Mice were sensitized with cholera toxin (CT) andpepsin gliadin (P-G) (1× per week for 3 weeks). During the experimentalperiod, mice were gavaged with Nepenthes extract-digested gliadin (3×per week for 3 weeks).

Treatment 2 (n=8):

Mice were sensitized with cholera toxin (CT) and pepsin gliadin (P-G)(1× per week for 3 weeks). During the experimental period, mice weregavaged with nepenthesin II-digested gliadin (3× per week for 3 weeks)

Results:

All 4 groups of mice were sensitized with pepsin-gliadin digest pluscholera toxin. Negative controls were left free of gliadin challengeafter sensitization. Positive controls and the treatment groups wereorally challenged with gliadin after sensitization. The difference inthe treated groups was that the gliadin challenge was pre-digested withNepenthes extract or nepenthesin II. In this way, the “negativecontrols” were not totally naïve of gliadin (since they were exposedduring sensitization phase), and thus mimicked the clinical situation ofa celiac patient going into remission while adhering to a gluten-freediet.

Clinical/Toxic Effects:

Overall appearance of the mice (movement, eye opening, grooming) wasevaluated. No ill effects were observed in any of the treatment orcontrol groups. Body weights were recorded throughout the experimentsand no weight loss was observed in any of the groups (FIG. 11).

Innate Immune Changes to Gliadin Challenge:

Immunohistochemistry for CD3+intraepithelial lymphocytes was performedon the intestines of mice from each treatment group (FIG. 12). This is aquick and early innate immune marker of intestinal gliadin exposure inthe model. Gliadin exposure resulted in increased IEL counts compared tonegative control mice and to mice exposed to gliadin that waspre-digested with Nepenthes extract or nepenthesin II (FIG. 13). Nodifferences in IEL counts were observed between Nepenthes extract andnepenthesin II treated groups.

Villus to Crypt Ratios:

Non-significant trends were observed for lower villus/crypt (V/C) ratiosin the positive control group (FIG. 14). Nepenthes extract andnepenthesin II treated groups had a trend for higher ratios compared tothe positive and negative controls.

Interpretation/Discussion:

A three-week challenge with gliadin pre-digested with Nepenthes extractor nepenthesin II was safe and did not induce short-term decreases inbody weight or any clinical adverse event in mice.

Oral gliadin challenges led to significant increases in small intestinalIEL counts in previously sensitized in mice. The IEL increase was notobserved in mice that were challenged with gliadin that had beenpre-digested with Nepenthes extract or nepenthesin II. This suggests alower luminal antigenicity of the gliadin treated with Nepenthes extractor nepenthesin II.

Reduction in V/C ratios was very mild in the positive control group.However, there were non-significant trends for higher V/C ratios in micethat were challenged with gliadin that was predigested with Nepenthesextract or nepenthesin II. Reduction in V/C ratios in this animal modelis moderate and varies with the duration and dose of the gliadinchallenge. The differences are more marked between positive and negativecontrols when the latter are completely naïve of gliadin/gluten(non-sensitized). It is believed that differences in V/C ratios usingpredigested Nepenthes extract or nepenthesin II in a more chronicsetting and/or compared to mice that are completely naïve of gliadin(non-sensitized) would be more pronounced.

Overall Conclusion:

The results show an effect of pre-digestion of gliadin with Nepenthesextract or nepenthesin II to reduce the antigenicity of the gliadinpeptides in the small intestinal tract of sensitized NOD/DQ8 mice.

Example 10 Gliadin Digestion by Neprosin

Crude neprosin extract was incubated with gliadin at pH 2.5 and theresulting peptide fragments analyzed by MS. The results are shown inFIGS. 15A and 15B (a dot [.] indicates a cleavage site). The proteinsequence coverage by the extract was 61%. Approximately 57% of thepotential proline (P) cleavage sites (C-terminal) in gliadin wereprocessed by the crude neprosin extract. Without being bound by theory,it is believed that at least a portion of the glutamine cleavage siteswere due to a small amount of contamination of the extract withnepenthesin proteins.

1. A method for attenuating or preventing intestinal inflammationarising from the presence of peptidic food antigens in a patient in needthereof, which method comprises administering to the patient aneffective amount of a pharmaceutical composition comprising an enzymeselected from the group consisting of nepenthesin I, nepenthesin II,neprosin, a variant thereof, and mixtures thereof, so as to cleave saidantigens into non-antigenic peptides, thereby attenuating or preventingsaid intestinal inflammation.
 2. The method of claim 1, wherein theintestinal inflammation is characterized by infiltration and/orproliferation of intraepithelial lymphocytes (IELs) in the intestine ofthe patient, and further wherein the infiltration and/or proliferationof IELs in the intestine is attenuated or prevented. 3-4. (canceled) 5.The method of claim 1, wherein the peptidic food antigens includepartially hydrolyzed gluten protein. 6-8. (canceled)
 9. The method ofclaim 1, wherein the patient suffers from a disease selected from thegroup consisting of gluten sensitivity, celiac disease, attentiondeficit hyperactivity disorder, autism, rheumatoid arthritis,fibromyalgia, nutrient malabsorption, and dermatitis herpetiformis. 10.The method of claim 1, wherein the inflammation is due to celiac diseaseaggravated by the presence of gluten.
 11. A method for attenuating orpreventing a manifestation of celiac disease arising from the presenceof antigenic partially hydrolyzed wheat protein in an intestine of apatient having celiac disease, which method comprises administering tothe patient an effective amount of an enzyme composition comprising anenzyme selected from the group consisting of nepenthesin I, nepenthesinII, neprosin, variant thereof, and a mixture thereof, so as to cleavesaid partially hydrolyzed wheat protein into non-antigenic peptides,thereby attenuating or preventing a manifestation of celiac disease. 12.The method of claim 11, wherein the partially hydrolyzed wheat proteinis a partially hydrolyzed gluten protein.
 13. The method of claim 1,wherein the pharmaceutical composition is a sustained-releaseformulation.
 14. The method of claim 1, wherein the pharmaceuticalcomposition comprises an extract of Nepenthes pitcher fluid.
 15. Themethod of claim 1, wherein at least one of nepenthesin I, nepenthesinII, neprosin, or variant thereof is a recombinant protein. 16.(canceled)
 17. The method of claim 1, wherein the pharmaceuticalcomposition is between about pH 5 and about pH
 8. 18. A method forattenuating or preventing intraepithelial lymphocytosis due to thepresence of peptidic food antigens in an intestine of a patient in needthereof, which method comprises administering to the patient aneffective amount of enzyme pharmaceutical composition comprising anenzyme selected from the group consisting of nepenthesin I, nepenthesinII, neprosin, and mixtures thereof, under conditions wherein saidantigens are degraded so as to attenuate or prevent intraepitheliallymphocytosis in the intestine. 19-24. (canceled)
 25. The method ofclaim 1, wherein the effective amount of the pharmaceutical compositionis between about 1 mg and about 1 g. 26-31. (canceled)
 32. A solidpharmaceutical composition comprising neprosin and a pharmaceuticallyacceptable buffering agent, wherein the neprosin is a protein comprisingan amino acid sequence having at least 90% sequence homology to theamino acid sequence of SEQ ID NO.: 1 and further wherein the bufferingagent maintains the composition at a pH of 6.5 or above prior toadministration, such that said composition is formulated for oraladministration.
 33. The pharmaceutical composition of claim 32, whereinthe amino acid sequence of the neprosin comprises an amino acid sequencehaving at least 90% sequence homology to the amino acid sequence of SEQID NO.: 1 without the signal sequence.
 34. The pharmaceuticalcomposition of claim 33, further comprising at least one additionalNepenthes enzyme or variant thereof.
 35. The pharmaceutical compositionof claim 34, wherein the at least one additional Nepenthes enzyme orvariant thereof is nepenthesin I, nepenthesin II, and/or a variantthereof.
 36. The pharmaceutical composition of claim 32, which is asustained release formulation. 37-39. (canceled)
 40. The pharmaceuticalcomposition of claim 34, wherein the neprosin and/or Nepenthes enzyme(s)comprises a propeptide.
 41. (canceled)
 42. A pharmaceutical formulationcomprising the composition of claim 32, wherein the neprosin is presentin multiple layers such that the neprosin is released continuously whilethe formulation is present in the stomach. 43-50. (canceled)